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The present book “Anatomical Techniques: New Era Tools in Health and Disease” is a compilation of various techniques from different sources by different authors just to facilitate the researchers to provide a comprehended resource material. The author during his early research days faced a lot of difficulties to explore various methods from different books/manuals which aided an extra burden to get available the material. These experiences made the vision to come true in the form of a comprehensive technique book. The book as a ready source material will prove a boon to the budding learners, trained researchers and established laboratory professionals. Biopsy, smears and FNAC are playing significant supportive role since ages. In the modern era, the clinicians and physicians glean a vast amount of information from laboratory tests for screening health status and disease diagnosis which ultimately pays towards better and effective treatment. These qualitative and quantitative techniques at micro and nano levels have created their pivotal spaces with no alternative replacements. All the techniques have been summarised in a simple way keeping in mind the better understanding of the students. The book has been organised in a sequence of techniques related to gross anatomy, plastination, histology, histochemistry, histoenzymology, scanning and transmission electronmicroscopy. The emerging new thrust areas like confocal microscopy, stem cells, PCR etc. have also been incorporated in the book. The book not only provides fundamental approach to hands on training on practical component but also provide theory component. The authors of the chapters have vast experience in their respective fields of specialisation. A due credit has been given to the original authors and reference books. This will aid in keeping alive their names, scientific contributions and legacy as most of the source materials are out of printing and on the verge of extinct. Sincere thanks to the publishing house for giving a shape in the form of a book to the scattered manuscripts

Teaching and research in anatomy is mainly based on cadaver dissections. A sound knowledge of anatomy is essential from the beginning of veterinary education and the knowledge obtained through dissection of animal body is an indispensable part of the veterinary science. History of embalming Embalming is the art and science of preserving the animal by treating with chemicals to forestall decomposition. Embalming destroys the colloid nature of the proteins and neutralizes the active centres of the molecules forming many cross linkages of adjacent protein molecules. Proteins are thus converted to high molecular inert solid materials that can no longer serve as food for bacteria or as a substrate for enzyme action. Embalming preserves the body from decay. The embalmed bodies are stored in tanks containing a fluid composed of formalin, phenol, thymol and rock salt in water in varying proportions. Claudius Galen (130-200 A.D) did dissections of animals such as the pig or monkey. Leonardo Da Vinci (1452-1519) a pioneer anatomist introduced anatomical drawings. During 15th-16th century the motive was to advance the techniques for the preservation of the dead and to study detailed anatomy by dissections. Andreas Vesalius (1514-1564) believed that dissection was necessary to find out how bodies worked. His lectures were based purely on his observations. He carried out dissection on human cadavers as well as animals to understand Anatomy. As an Anatomist he represents the end of the middle ages obscurantism and the beginning of modern Anatomy and scientific thinking. He is considered the father of modern Anatomy. In 17th century Antonie Van Leeuwenhoek (1632-1723) opened up new horizons

The study of Veterinary Anatomy is an integral part of Veterinary Science as it evolved as a keystone of veterinary education through ages. Osteology (study of bones) is the essential part of the anatomy teaching curriculum. The bones are unsurpassed in the ability to provide the information on the three dimensional form and architecture of the body. It also helps in understanding the disposition and relations of soft tissues along with the course of neurovascular structures in that region. For osteological study, the bones are collected from the carcasses and are duly processed. Bone preparation involves soft tissue removal, cleaning and bleaching of bones and joints, articulation and labeling. The skin, visceral organs and the maximum possible amount of flesh adhering to the bones are removed by knife. Such bones are called wet bones. The whole skeleton is disarticulated; however, the pedal bones are left with the hoof. In case of skull, the mandible is dismembered and the mouth parts are removed. The tongue is carefully sliced to locate the hyoid bone. It is separated at the hyoid process of the petrous temporal bone. The brain is lacerated and goosed out of cranial cavity with a stick through the foramen magnum. The eyeball is carefully extirpated without any injury to the orbit. The nasal cavity is left untouched with the turbinates and cartilages intact. Following steps are involved in processing of bones and preparation of skeleton. A. Maceration B. Degreasing C. Bleaching D. Varnish E. Categorization of bones F. Preparation of articulated skeleton

Animals experience the effects of force in everyday life. For example, the animal body exerts a force on the body and to the objects during various agricultural operations. Furthermore, the gravitational force exerts continuously, for instance while sitting, walking, etc. All parts of the body in one way or the other are loaded by forces. The bones provide rigidity to the body and can sustain high loads. The skin is resistant to force. The cardiovascular system is continuously loaded dynamically owing to the pulsating blood pressure. The bladder is loaded and stretched when it fills up. The intervertebral discs serve as flexible force- transmitting media that give the spine its flexibility. Beside force, animals are using levers all the time in their daily life to increase the ‘force’ that is applied to some object. Animal soft tissues are complex materials that can exhibit nonlinear, time dependent, inhomogeneous, and anisotropic behaviors. The mechanical responses are reflected in all levels of organism (molecules, organelle and body levels). Several biological activities are under the action of forces, which even govern the gene expression. The study dealing with the forces and their effects on living systems is the Biomechanics. Biomechanics applies the laws and the techniques of mechanics in the study of biological systems and phenomena. Indeed, mechanics provides the tools for:

Biometry is the measurement of various grossly visible dimensions of organs and their application in statistical analysis to generate biological data. Various biometrical parameters used for different organs The biometry of different organs is conducted with the help of non-stretchable thread, Vernier callipers, measuring scale, protector, glass beaker of various sizes, calibrated measuring cylinders, etc. While conducting biometry, it should be kept in mind that the way or the procedure for conducting biometry of different organs varies due to variation in shapes of the same and their positions. Some of the biometrical parameters of different organs and bones are summarized below. 1. Length: May be used to measure the distance between the highest points along the length of any bone or organ. Usually expressed in cm. Non-stretchable thread, Vernier callipers and measuring scale are used for this purpose. 2. Breadth: May be used to measure the distance between the highest points along the breadth of any bone or organ. Usually expressed in cm. Non-stretchable thread, Vernier callipers and measuring scale are used for this purpose. 3. Thickness: May be used to measure the distance between the highest points in regard to the thickness of any bone or organ. Usually expressed in cm. Non-stretchable thread, Vernier callipers and measuring scale are used for this purpose. 4. Volume: It is measures by water displacement of any organ. Usually smaller organs are used. The fresh organ is dipped into a calibrated

The whole brain or its slices could be stained for differentiation of the areas of gray and white matter for better understanding of macroscopic neuroanatomy. Various researchers have developed the methods of staining of either the whole brain or its slices. The review of all these methods is being detailed below. These would definitely help the teachers and students to teach and grasp the macroscopic details of central nervous system viz. nuclei, laminae, fascicule, capsule and other structures with little or no magnification. Staining of Whole Brain and Sections from its Slices (Hewitt, 1959) Fixation of whole brain in either 70% alcohol or 10% formaldehyde without removal of membranes and blood vessels. Protocol 1. The collected whole brain is immersed in ten volumes of staining fluid. 2. The staining fluid can be resaturated, when ever it becomes paler and clearer in colour, then it can be used in smaller volume in proportion to the volume of brain for the next staining. 3. Complete staining is assessed by uniform red colouration of the brain. A cats’ brain requires at least 1 month and the human brain not less than 6 months. The time required is governed by size of the brain. The brain stem is cut of about 5 mm thickness to examine the completion of the staining. 4. The stained brain is immersed in ten volumes of 70% alcohol for differentiation.

Taxidermy The word ‘Taxidermy’ is derived from the Greek taxis meaning “fixing” or arrangement and derma meaning ‘skin’. It is possible to perform taxidermy of mammals, birds, fishes, reptiles and amphibians and procedure to perform taxidermy is also variable accordingly. In this case we will see the procedure applied to prepare a big cat (Tiger, lion, leopard etc.). Skinning To skin an animal make an inclusion from the chin along the belly to tip of tail, Then from paw to paw across the chest, and deal similarly with the hind legs. If they are done haphazard, The appearance of the skin will be completely wrecked. Place the carcass on its back and make an incision through the chin and carry it along the centre of the throat to the chest. There is a whorl of hair which runs from the chest along the belly to the vent, then carry the incision along from the vent to tip of tail. Fore- legs, after cutting through the ball of the pad the incision must be made exactly through the center of the white till it reaches the incision which is made from chin to tip of tail, the other foreleg is treated in the same way, both these incision should meet on the chest, and not end one above the other, Hind-legs after cutting through the ball of the pad. The incision runs behind the leg to end of the calcaneum, then turn inside the thigh. The hind leg, of course must be done in the same way. It will be better to mark with charcoal on proposed incision lines before the skinning carried out. After the pads have been skinned and turned back, also inside of the thigh and belly, there is not much need for the knife as the skin ought to be removed by what butchers term ‘ fisting ‘. Get someone to hold the leg to keep the carcass rigid, when the skinners pull the skin taut with his left hand and, with his right first clenched, process vigorously on the skin close to the

Veterinary anatomy is a backbone as well as basic discipline in the veterinary sciences at large. The new entrants of the first professional year of B.V.Sc. & A.H. get more exposure in gross anatomy, histo- architect models and developmental anatomy of their current curriculum in our country. Laboratory demonstration plays a vital role module in virtual teaching and learning program of their better understanding of the subject. The visualization and memorization is the basic requirement for thorough understanding of the anatomy of the individual in medical sciences. The various method of teaching have been debated because it has own limitations. To improve the anatomical understanding the traditional way of using cadavers but with current scenario of restrictive laws which implemented by Veterinary Council of India (VCI) and other organizations viz, SPCA, PFA, CPCSEA/IAEC etc. The scarcity of specimens which are invited the other alternative way of keeping and preserving the specimens for laboratory teaching. Under the unusual circumstances, the normal conventional method of cadaver/specimen/organ preservation should be replaced by the advanced technologies for easy and interesting to learn. In the traditional curriculum method of anatomical specimens taught and learned by formalin fixed cadavers/specimens/tissues. Since, the conventional method creates health problem due to extensive use of formalin as preservative chemical for cadaver preservation. This method of preservation causes wet slippery tissues, respiratory irritation, topical allergic reactions, and students should wear gloves to protect themselves from the fixative like formaldehyde (act as a potential carcinogenic agent). However, the anatomists have been challenged for an alternative laboratory teaching aids in near future. In recent past, the alternative method has developed teaching materials by the innovative novel techniques like Plastination, corrosion cast, pneumatic air dried specimen etc.

1. Fix the organ or the animal in 5-10 % formalin preferably neutral buffered formalin. If the organ is an isolated thoracic limb, for example, then cannulate the axillary artery and perfuse the formalin. I find sometimes it is advantageous to pump some buffered saline solution to flush out any blood before pumping in the formalin. Organs, such as the heart, could be nicely flushed with water through major blood vessels, and immersed in formalin to fix. The same can be done with liver, kidney, etc. It takes a week for fixing a large structure (e.g., 10 kg) by immersing in formalin. 2. Dissect the specimen as needed. Remove all fat and clean as much as possible so the specimen looks nice. 3. If the specimen is dark brown, it can be lightened by leaving 1-2 days in 5% hydrogen peroxide. Check the specimen 1-2 times during the day to make sure desired light tone is achieved. Leaving it too long in the hydrogen peroxide may result in tissue degradation. 4. Drain the specimen well, and dehydrate in acetone. The dehydration should be thorough. To achieve this, use 100% acetone, at least 2-3 changes. The acetone can be recycled by repeated distillation. Acetone tends to evaporate, so use a container that has a tight lid. Acetone is also flammable, so use required precautions when handling acetone. If concentration of acetone remains up to 99% even after leaving organ for 4-5 days it is sure that dehydration is complete. If you are using any thin sheets of organ or Brain for dehydration keep slice between glass plates and then dehydrate. 5. Immerse the specimen in glycerin. Buy industrial glycerin, which may be cheaper. It may take 2-3 weeks for the glycerin to permeate into the tissues.

It is an inherent, distinctive and implicit human nature to preserve and care for objects that seem unique, rare and adorable. This instinctive desire of the human mind must have invented the concept of museum. The word museum derives from the Greek word “Mousein” which mean “Seat of the Muses”. The muses are the divine patron of arts in Greek Mythology. A museum by definition is “An institution that houses and cares for a collection of artifacts and other objects of scientific, artistic or historical importance and makes them available for public viewing through exhibitions that may be permanent or temporary (Alexander and Alexander 2008; Kamath et al., 2014). The anatomy museum comes under the category of science museum. Anatomy museum define as a place where bones, models, charts wet specimen of permanent value are kept and displayed. It has to be designed to function as an institute where active leaning can take place (Kamath et al., 2016). The museums now witnessed a transition from an institute housing a collection of models and artistic sketches to one housing formalin preserved specimens, corrosion casts and plastinates. The contemporary museums have sophisticated interiors, display, lightings and use computerized catalogues and audio-visual aids to teach anatomy. History The concept of anatomy museum was first conceived by the Edinburg Surgeons in 1763 A.D. A cabinet with anatomical picture books and few specimens was first prepared and named as cabinet of curiosities. Early museums housing a simple collection of wood, clay, wax or ivory models of anatomical specimens. The discovery of formalin in 1859 by Alexander Mikhailovich Butlerov was a great milestone in anatomical history and changed the, manner of anatomical specimen presentation. Later on discovery of different methods significantly make transition in the nature of collection exhibited in anatomy museum.

Accurate estimation of embryonic/foetal age play vital role in the developmental anatomy research related to morphogenesis and histogenesis of an organ or system. The observations and recordings of normal developmental features of embryos/ foetuses at successive stages of gestationare very important for studying functional development of an organ in anatomical research. The documentation of normal embryonic and fetal growth can serve as a guide for understanding the consequences of harmful influences at various stages of gestation (Evans and Sack, 1973). Post-mortem studies have provided valuable information on fetal development, identifying stages such as implantation, appearance of forelimb bud, tail development and hind limb development (Alberto et al., 2013). The normal development of external features of animals has been described by Evans and sack (1973), however, subjective assessment of foetal age from the external features alone is not much reliable, although it may be useful adjunct to more precise measurements (Richardson et al., 1990). For accurate estimation of developmental age, fetal measurements are needed which show a consistently rapid rate of growth and which are least variable at a given age (Richardson et al., 1990), however, these parameters are affected by individual variation and by breed differences within a species (Greenwood et al., 2005). A number of methods have been discussed by many workers for estimation of age of foetus in animals. The method mainly focus on foetal biometry recorded either inside uterus during normal gestation process or after taking out foetus during post-mortem examination. In-utero foetal biometry allows estimating gestational age and time of parturition by ultrasonographic methods (Manning

Routine paraffin wax embedding technique can be conveniently discussed under the following five headings: Stages of tissue processing 1. Fixation: Stabilizes and hardens tissue with minimal distortions of cells 2. Dehydration: Removal of water and fixative from the tissue. 3. Clearing: Removal of dehydrating solutions, making the tissue components receptive to the infiltration medium. 4. Infiltrating: Permeating the tissue with a support medium. 5. Embedding: Orienting the tissue sample in a support medium and allowing it to solidify. Fixation-A It is essential that tissues be fixed as soon as possible after death or removal from the body, and for this reason screw-capped specimen jars containing appropriate fixatives should be permanently kept wherever tissues for histological examination are taken regularly, for example in the operating theatre, the post- mortem room, or the animal house. The amount of fluid in the jars should be 15-20 times the bulk of the tissue to be fixed. Care should be taken to ensure early despatch of the specimens to the histology laboratory in order to avoid over-fixation in intolerant fixatives. Tissues selected for sectioning should be sufficiently thin to be adequately fixed throughout in a reasonable time. The overall bulk of the tissue determines the volume of fixative required; the thickness will determine the speed of fixation.

Sectioning of tissue is carried out by using an instrument called microtome and the procedure is known as microtomy. Most commonly used microtome is HYPERLINK http://paramedicsworld.com/microtome/ rotary microtome which allows the perfect sectioning of the paraffin-embedded tissue specimens. For histological purposes, section cutting is done at the thickness of 4-6 microns whereas for histochemical staining techniques, the thickness of sections may be increased. Pre-steps involved in tissue section cutting 1. Ensure that microtome is placed on an even surface so that it will not be shaken during the procedure of section cutting 2. Ensure that Knob for the rotating handle of microtome is locked to avoid cutting the fingers of handler of microtome while fixing the blade in its holder 3. Ensure that paraffin shavings from the previous section cutting were removed from the various parts of microtome for smooth functioning of microtome 4. Thermostatically controlled water bath or tissue flotation bath should be kept ready with filled water to heat 5. Ice cubes should be kept ready in the refrigerator. Success of sectioning can be greatly improved by cooling the wax blocks before subjecting them to cut. To properly cool the blocks, placing them on a piece of aluminum foil on a levelled tray of ice for at least five minutes before sectioning is preferable. Chilled paraffin wax is harder and in general it allows thinner and higher quality sections .Cooling both the tissue and wax giving them a similar consistency, in addition to a smaller amount of water will be absorbed into the tissue and making sectioning easier.

A useful method for quickly preparing tissue slides for microscopic analysis is the frozen section technique. There are numerous clinical and research contexts where the frozen section technique is employed. For quick intraoperative diagnosis in histopathology, frozen sections are frequently utilized, giving our surgical colleagues direction. To ascertain the extent of excision required to remove a skin tumour, the surgeon doing Mohs Micrographic Surgery depends solely on the frozen sections. The frozen section technique is used in many scientific applications to create microscopic slides using a variety of complex morphologic, immunohistochemical, and molecular techniques. There are several uses for frozen sections, including rapid diagnosis, immunohistochemistry, enzyme histochemistry, lipid detection, cancer margin research, and more. Making frozen section slides is a difficult technical procedure that calls for the mastery of advanced technical abilities in addition to knowledge of the pathology, microanatomy, and histology of the tissues under investigation. The outcomes will depend on our capacity to produce a high-quality preparation, whether it is utilized for research or intraoperative consulting. Dr. Louis B. Wilson’s 1905 description served as the basis for the frozen section technique used in medical labs today. At the request of surgeon Dr. William Mayo, one of the founders of the Mayo Clinic, Wilson devised the procedure based on previous studies. A frozen section was also used in earlier reports by Dr. Thomas S. Cullen of Johns Hopkins Hospital in Baltimore, but only after formalin fixation. Dr. William Welch, a pathologist at Hopkins, also tried Cullen’s technique, but there were no negative clinical effects. Wilson is therefore widely acknowledged as having really invented the procedure.

Types of bones Bones forming the animal skeleton can be classified into two types such as compact bone (cortical) and spongy bone (trabecular / cancellous). The compact bone is found in diaphysis or shafts of all the long bones of forelimbs, hindlimbs and outer surfaces of the flat bones of skull and ribs. In contrast, the spongy or trabecular bone is found in epiphysis or extremities around the bone marrow cavities of long bones and centre of flat bones and vertebrae. As the name implies, the compact bone is solid and hard due to the arrangement of special collagen in the bone (Type I) forming the bands or lamellae parallel to each other surrounded by a cement of proteoglycan ground substance deposited with minerals. Whereas, the spongy bone is made up of meshwork of bone strands running in all directions arranged as trabeculae adapted for weight bearing ability in heads and condyles of long bones. Structure of bone tissue The bone consists of bone cells, extracellular organic matrix made of collagen fibres and minerals. The cells of bone are osteoblasts, osteocytes and osteoclasts. Osteoblasts are low columnar to squamous cells responsible for active synthesis and mineralization of bone matrix found mainly in the periosteum and endosteum. Osteocytes are responsible for structure of the bone and calcium homeostasis found in the mature bone. It is located in the lacunae with extensive cytoplasmic processes running through canaliculi with in the bone matrix to make communication between adjacent osteocytes. Osteoclasts are multinucleated (15-30 nuclei per cell) larger cells responsible for bone resorption and remodelling. The secretion of these cells composed of acid and lysosomal enzymes responsible for erosion of bone tissue thereby formation of Howship’s lacunae (Frappier and Eurell, 2006).

The hematoxylin and eosin stain are most widely used histological stain. Its popularity lies in its simple staining procedure and ability to demonstrate most tissue structures. Hematoxylin, also called natural black is a compound extracted from heartwood of the logwood tree (Haematoxylum campechianum) with a chemical formula of C16H14O6. This naturally derived dye has been used as a histologic stain, ink and as a dye in the textile and leather industry. Hematoxylin, a natural dye was first used in about 1863. Hematoxylin itself is not a stain; it is its oxidation product, hematein. This process known as “ripening” takes several days or weeks unless it is hastened by the addition of an oxidizing agent. So the active coloring agent hematein can be produced from hematoxylin in two ways: A. Natural oxidation by exposure to light and air. B. Chemical oxidation: Sodium iodate is the most common oxidizing agent for this purpose, although there are others, such as potassium permanganate, iodine, bleach and mercuric oxide. It is now strongly recommended that mercuric oxide not be used for this oxidation as it eventually makes its way into the environment as a poison. As the hematoxylin dye has little affinity for the tissue when used alone, it is usually used in conjunction with a mordant, therefore can be arbitrarily classified based on which mordant is used: 1. Alum hematoxylin 2. Iron hematoxylin 3. Tungsten hematoxylin

Fixation is the first and utmost important step to take good histology of eye ball. The eye is a composite organ made up of layers of different consistencies that tend to separate during fixation and sectioning (Drury and Wallington, 1980). Fixation (Davidson’s fluid) Davidson’s fluid is an excellent fixative for fixation of whole eye ball but conventional 10% formalin causes artificial cellular shrinkage and poor cellular and nuclear resolution of the retina. Formaldehyde penetrates tissue well but takes more time because sclera stands as physical barriers that protects the retina and inhibit the penetration of fixative. Davidson’s fixative is acetic acid, alcohol- formalin based fixative in which alcohol denatured the protein by breaking hydrogen bonds and disturbing their tertiary structure and acetic acid increase the penetration. It has been advocated and widely used for the preservation of eye ball, maintaining retinal attachment during fixation and processing and providing better preservation of the retinal nuclear layer and sensory specialization of the rods and cones.

Van-Gieson Stain for Collagen Fibers Tissues fix in 10% neutral buffered formalin and process for routine paraffin sections Protocol 1) Deparaffinize sections and hydrate to water through descending grades of alcohol. 2) Remove mercury crystals if tissue fixed in Zenker’s or Helly’s fluid. 3) Stain in iron haemotoxylin solution for 2 to 3 minutes. 4) Wash in running water for 10-15 minutes. 5) Rinse in distilled water. 6) Counter stain in Van Gieson’ssolution for 2-3 minutes. 7) Two to three dips in 96% alcohol. 8) Dehydrate through absolute alcohol and clear in xylene. 9) Mount with D.P.X mountant. Observations: Red : Collagen fibers Yellow : Muscles, RBC and cornified epithelium Blue : Nuclei

McManus’ Method for Glycogen (PAS) Tissues fix in 10% neutral buffered formalin and process for routine paraffin sections Protocol 1) Deparaffinise the paraffin sections with two changes (10 minutes each) of xylene. 2) Descending grades of ethanol from absolute to 50% solution (2 minutes each). 3) Hydrate the sections with distilled water 5 minutes. 4) 0.5% Periodic acid solution for 5-6 minutes. 5) Rinsing in distilled water. 6) Schiff’s’s reagent or Coleman’s Feulgen solution for 10-15 minutes. 7) Washing in running tap water for minimum 10 minutes until pink colour develops. 8) Counter stain with Harris’ hematoxylin for 2-3 minutes to stain nuclei. 9) Washing in running water for 2-3 minutes. 10) Quick dip in ammonia water to develop blue colour of the nuclei. 11) Washing in running tap water for 5 minutes. 12) Dehydrate in ascending grades of 90% to absolute ethanol (one minute each). 13) Clearing in xylene, two changes of 30 minutes each.

A protein that is deposited in various tissues of body called amyloid. Amyloidosis is a disorder of protein folding, in which normally soluble proteins accumulate in the tissues as abnormal insoluble fibrils (or filaments), thus disrupting their function The deposited proteins, generically known as amyloid, damage the structure and function of the tissues and so cause serious disease which is often fatal when it affects major organs. There are several techniques to observe the amyloid which is as follows. Highman’s Congo Red Technique Tissues fix in 10% neutral buffered formalin and process for routine paraffin sections The formal saline is a better choice Protocol 1) Sections to water, removing pigment where necessary. 2) Stain in Congo red solution, 5 minutes. 3) Differentiate with the alcoholic potassium hydroxide solution, 3-10 seconds. 4) Wash in water, stain nuclei in alum hematoxylin and differentiate blue. 5) Dehydrate, clear, and mount. Observations Red : Amyloid, elastic fibers, eosinophil granules: Blue : Nuclei Chemical reagents Immerse in alkaline sodium chloride solution for 20 minutes.

Lipids are naturally occurring fat like substances insoluble in water but are soluble in organic solvents like alcohol, ether, and chloroform etc. Some of the phospholipids are even soluble in water. All the lipids do not resemble fats. Lipids are an important component of living cells. Together with carbohydrates and proteins, lipids are the main constituents of animal cells. Lipids include fatty acids, neutral fats, waxes and steroids (like cortisone). Compound lipids (lipids complexed with another type of chemical compound) comprise the lipoproteins, glycolipids and phospholipids. Properties of lipids Lipids are non-polar molecules a family of organic compounds, composed of fats and oils yield high energy and are responsible for different functions within the body. It is stored in the adipose tissue of the body. Lipids are a heterogeneous group of compounds, mainly composed of hydrocarbon chains. Lipids are energy-rich organic molecules, which provide energy for different life processes. Lipids are a class of compounds distinguished by their insolubility in water and solubility in nonpolar solvents. It is important constituent of cell membrane in biological systems, a mechanical barrier that divides a cell from the external environment. Most of the lipids or fats are demonstrated by using fat soluble dyes. The lipid soluble dyes have greater affinity for fats, lipids, lipoproteins and triglycerides. The fat soluble dyes are:

The Millon Reaction Tissues fixation in 10% neutral buffered formalin solution Protocol 1) Deparaffinise the paraffin sections with two changes (10 minutes each) of xylene. 2) Descending grades of alcohol from absolute to 50% solution (2 minutes each). 3) Hydrate the sections with distilled water for 5 minutes. 4) Transfer the sections to a small beaker with the reagent and gently boil. 5) Stop the heating process and let the solution cool to room temperature. 6) Remove the sections and rinse them three times in distilled water (2 minutes each). 7) Dehydrate in ascending grades of 90% to absolute alcohol (one minute each). 8) Clear in xylene, two changes of 30 minutes each. 9) Mount glass cover slip with DPX.

Schultz Method for Cholesterol Frozen sections; cold calcium-cadmium-formalin fixed sections Protocol 1) Thoroughly wash the sections in distilled water for 24 hours, changing the water several times. 2) Incubate the sections in 2.5% ferric ammonium sulphate solution at 37oC for 7 hours. 3) Wash the sections three times in fresh acetate buffer, allowing 1 hour for each wash. 4) Rinse briefly in distilled water. 5) Immerse in 5% formalin for 10 minutes. 6) Mount the sections onto slides, blotting the edges to remove excess water. Do not let the sections dry out. 7) Place a drop of a 1:1 mixture of sulphuric and acetic acids on a coverslip. Invert the slide and lower it onto the coverslip. Then turn the slide rightside up and gently press the coverslip to flatten the section. Hold the coverslip by its corners and gently oscillate it a few times to ensure even spreading.

Bielschowsky’s Method for Axis Cylinders and Dendrites Tissues fix in 10% neutral buffered formalin and process for routine paraffin sections Protocol: 1) Deparaffinise the paraffin sections with two changes (10 minutes each) of xylene. 2) Descending grades of ethanol from absolute to 50% solution (2 minutes each). 3) Hydrate the sections with distilled water 5 minutes. 4) 2% Silver nitrate solution for 48 hours, in the dark. 5) Rinse quickly in double distilled water. 6) Ammoniacal silver solution 10-20 minutes or until sections turn a deep brown. 7) Rinse in distilled water. 8) Reduce in 20% formalin solution for 5 minutes. Sections appear a dark brownish black. 9) Rinse thoroughly in distilled water. 10) Tone in gold chloride solution for 1 hour. Sections will be a reddish violet colour. 11) Rinse thoroughly in distilled water. 12) Sodium thiosulfate solution for 1 minute. 13) Wash in tap water. 14) Dehydrate in ascending grades of 90% to absolute ethanol (one minute each).

A large population of endocrine cells secrete the same chemical messengers (bio-active animals and regulatory peptides) as do the neurons and therefore these endocrine dells are referred as “Paraneurons”. However these messengers are not used as neurotransmitters, rather they are released into blood circulation and interstitial fluid as hormones/chemical mediators to act on the target cells in endocrine or paracrine manner. This subset of endocrine cells forms the “Neuroendocrine system” and its synonyms are the “diffuse neuroendocrine system (DNES)”, “APUD (amine precursor uptake and decarboxylation) cell system” and the “regulatory peptide secreting cell system”. The DNES consists of cell populations scattered throughout the body either to form a coherent tissue in some organs (e.g. anterior pituitary, pancreatic islets, adrenal medulla) or to form a diffuse scatter of single cells or group of 2-3 cells within the stroma of the other organs (e.g. C cells of thyroid, intra-epithelial endocrine cells of the gut, airway and urinogenital system, chief cells of parathyroid, glomus cells of carotid body, melanocytes of skin, some cells of pineal gland and placenta, modified cardiac myocytes, hypothalamic neurons synthesizing oxytocin and vasopressin as well as neurons synthesizing releasing factors controlling the secretion of hormones by the adenohyposis etc.). These cells synthesize and store the hormones in their secretion granules as neurosecretory granules which have characteristic ultrastructural morphology. The cells of the DNES can be identified by: (i) the ultrastructural morphology of the cells and their secretion granules, (ii) the empirical histochemical staining techniques which stain the neurosecretory granules, (iii) the novel current day, immunohistochemical techniques, or more aptly (iv) a combination of

Gomori’s Method for Iron The major deposits of iron in mammals are in the red blood cells as hemoglobin and in the phagocytes of the reticulo-endothelia system as ferritin and haemosiderin. It is essential mineral and an important component of proteins, involved in oxygen transport, oxygen storage in muscle tissues and oxidation of nutrients in the mitochondria. Enzymes of the electron transport chain, cytochromeoxidase, ferredoxin, myeloperoxidase, catalase and the cytochrome P450 enzyme also requires iron as co-factors. Tissues fix in 10% neutral buffered formalin or absolute alcohol and process for routine paraffin sections Protocol 1) Deparaffinise the paraffin sections with two changes (10 minutes each) of xylene. 2) Descending grades of ethanol from absolute to 50% solution (2 minutes each). 3) Hydrate the sections with water 5 minutes. 4) Hydrochloric acid-Potassium ferrocyanide solution for 30 minutes. 5) Rinse thoroughly in distilled water. 6) Counter stain in nuclear fast red solution for 5 minutes. 7) Rinse in distilled water. 8) Dehydrate in ascending grades of 90% to absolute ethanol (one minute each). 9) Clearing in xylene, two changes of 30 minutes each

Unna’s Method for Mast Cells Tissues fix in 10% neutral buffered formalin and process for routine paraffin sections Protocol 1) Deparaffinise the paraffin sections with two changes (10 minutes each) of xylene. 2) Descending grades of ethanol from absolute to 50% solution (2 minutes each). 3) Hydrate the sections with distilled water 5 minutes. 4) Polychrome methylene blue solution for 10 minutes. 5) Rinsing in distilled water. 6) Differentiate in glycerin-ether solution for 30 seconds to 1 minute. 7) Washing in water for 2-3 minutes. 8) Blotting with filter paper. 9) Dehydrate quickly in 100% ethanol. 10) Clearing in xylene, two changes of 30 minutes each. 11) Mount glass cover slip with DPX.

Pituitary Herlant (1960) Pituitary Stain I Tissues fix in 10% neutral buffered formalin or Zenker formol and process for routine paraffin sections Protocol 1) Deparaffinise the paraffin sections with two changes (10 minutes each) of xylene. 2) Descending grades of ethanol from absolute to 50% solution (2 minutes each). 3) Hydrate the sections with water 5 minutes (remove HgCl2 if present). 4) Stain in erythrosine for 5 minutes. 5) Rinse briefly in distilled water. 6) Stain in Mallory II for 5-10 minutes. 7) Rinse briefly in distilled water. 8) Stain in acid Alizarine blue for 5-10 minutes. 9) Rinse briefly in distilled water. 10) Treat with phosphormolybdic acid for 5-10 minutes. 11) Rinse briefly in distilled water. 12) Differentiate in 70% ethanol

May-Grunwald-Giemsa Stain (MGG) Fix slide in absolute methanol Protocol 1) Fix the slides with May-Grunwald reagent (15 drops) for three minutes. 2) Without removing the reagent, the same amount of distilled water is poured on the slide and allowed to stand for three more minutes. 3) The dilute May-Grunwald reagent is poured off and the slide is immersed for 8-12 minutes in a watch glass containing Giemsa reagent (15 drops of concentrated reagent are used for each 10 ml. distilled water). The slides should be immersed upside down in the solution to prevent the precipitate formed in the staining mixture from adhering to the preparation. 4) The slides are washed with tap water and dried in air or with filter paper. The distilled water must be neutral or otherwise buffered. With bone marrow or lymph node aspirates a longer staining time is recommended on account of the greater cell concentration. We recommend controlling the staining time with the microscope.

Stains are inevitable in histology to impart variable colours to different tissue components. In this domain, hematoxylin (H) and eosin (E) dyes are the dominant stains. According to an estimate nearly 2.5 million slides are stained with H & E each day, reflecting its enormous use in histology laboratories. Hematoxylin is a natural dye, extracted from bark of logwood (Haematoxylon campechianum), whereas eosin is a synthetic dye. The journey of using natural dyes in histology began with A. Leeuwenhoek (1673) who used “saffron”, an extract of saffron crocus. It continued until the systematic studies of C Weigert, J Gerlach, P Ehrlich and H Gierke to the second half of the 19th century and then this filed became subdued by the emerging era of synthetic dyes for their effective tinct. Today most of the stains used in histology are the synthetic dyes. Often, these dyes need further treatment with chemicals to formulate the final working solutions. Synthetic dyes are efficient, but may pose serious health hazards. The awareness on such hazards even has gone to an extent of withdrawal of some of the dyes from histological techniques. Frequent exposure to these chemicals constantly affects the health of the laboratory technicians, pathologist and mainly those working in the laboratory. Therefore, an era has triggered on the current day, to seek for use of the natural herbal dyes in histology. Despite various shortcomings with use of natural herbal stains, they can still be sought as an analogous to the conventional stains. The concern for safety, cost-effective, eco-friendly, biodegradability and local availability is driving the quest to explore the abundant local plant resources for this purpose. The precise research on various parts of the plants such as stems, leaves, flowers, fruits, roots and seeds used for dye extraction in our laboratory to stain the cell cytoplasm replacing the conventional eosin stain are chronicled in this documentation.

Enzyme histochemistry constitutes a link between biochemistry and morphology (Hardonk et al., 1976) and provides important information complementary to conventional histology, immunohistochemistry and molecular pathology (Meier Ruge and Bruder 2008). It helps to detect any metabolic changes in tissues and thus can aid in disease diagnosis. It is based on the principal that, the substrates added to tissue are metabolized by enzyme and the reaction is visualized by an insoluble dye product. It is a very sensitive technique that detects even an early metabolic imbalance in the tissue. For example, alkaline phosphatase activity represents tissue barrier functions in brain capillaries, duodenal enterocyte and proximal kidney tubule brush borders and a decrease in enzyme activity will indicate some functional impairment. Similarly, an increase in lysosomal acid phosphatase activity is an early marker of ischemic tissue lesions and acetylcholinesterase enzyme histochemistry has been used for the diagnosis of Hirschsprung disease (Meier Ruge and Bruder, 2008). ATPase methods are used in combination to distinguish between type1 and type 2 muscle fibres and NADH diaphorase is used to detect very minor or early structural abnormality in the sarcoplasmic reticulum network of the fiber, as well as mitochondrial abnormalities eg. Mitochondrial myopathies (Sargaiyan and Bansal, 2014). It has been clearly shown that tumor tissue with high glycolytic activity is particularly resistant to radiotherapy (Mathupala et al., 2007). Enzyme histochemistry for dehydrogenase provides a quick, efficient and inexpensive orientation on the actual energy metabolism of a particular tumor and thus aid in therapeutic strategy (Nakagawa et al., 2002). For the histoenzymic localization, fresh frozen unfixed tissues preferably 2 to 5 mm square size should be collected immediately after death/slaughtering of animals. Fixation in formalin will inactivate most of enzymes but formalin fixation is also employed to stop enzyme activity of lactic dehydrogenase and

Immunohistochemistry (IHC) is a vital research technique that employs labeled antibodies to identify and locate antigens in tissue sections. It has recently gained widespread popularity in anatomic pathology research globally. This method combines immunological, histological, and biochemical approaches to detect specific antigens using antibodies designed for those antigens. Antigen-antibody reactions are visualized through various methods under a microscope. When this technique is applied to individual cells or involves labeling at the electron microscopic level (using the immunogold technique), it is called immunocytochemistry (ICC). The overall process of performing immunohistochemistry or immunocytochemistry is known as ‘immunolabeling.’ Basic Principle of Immunohistochemistry Antibodies are crucial in triggering an immune response by precisely recognizing and binding to foreign molecules. This specific interaction between an antibody and an antigen leads to the formation of an antibodyantigen complex, which is fundamental to immune reactions. This principle forms the cornerstone of immunohistochemistry.

Lectins are sugar-binding proteins or glycoprotein of non-immune origin. In 1954 William C. Boyd alone and then together with Elizabeth Shapleigh introduced term ‘lectin’ from the Latin word lego- ‘chosen’ (from the verb legere ‘to choose’ or ‘pick out’).The earliest description of a lectin is believed to have been given by Peter Hermann Stillmark in his doctoral thesis presented in 1888 to the University of Dorpat. Stillmark isolated ricin, an extremely toxic hemagglutinin, from seeds of the castor plant (Ricinus communis). History Lectins, although first discovered more than 100 years ago in plants, and defined by their ability to selectively recognize specific carbohydrate structures, are ubiquitous in living organisms and throughout nature. Long before a deeper understanding of their numerous biological functions, the plant lectins, also known as phytohemagglutinins, were noted for their particularly high specificity for foreign HYPERLINK https://en.wikipedia.org/wiki/Glycoconjugate glycoconjugates (e.g. those of fungi, invertebrates, and animals) and used in biomedicine for blood cell testing and in biochemistry for fractionation. Hundreds of lectins of plant and animal origin have been described, and the list continues to increase. The first lectin to be purified on a large scale and available on a commercial basis was concanavalin A, which is now the mostused lectin for characterization and purification of sugar-containing molecules and cellular structures. The legume lectins are probably the most well-studied lectins.

Scanning electron-microscopes are available nowadays which operate at high vacuum as well as low vacuum mode. Low vacuum mode allows viewing of wet samples, but compromise on very high resolution and high magnification images. In high vacuum mode the samples must be absolutely dry and free of any organic contaminants to avoid gas molecules interfering with both the primary electron beam and the secondary or backscattered electrons emitted from the sample. Biological specimens which are largely composed of water, necessitate additional preparatory steps to ensure that structure of the organism is retained in its natural form. Sample preparation is essential in scanning electron microscopy. Improper sample preparations can undermine the quality of results and lead to false conclusions. SEM, unlike TEM, allows the processing of larger sized organisms and tissues. For Biological samples the smallest representative sample size should be used as the microscope’s detection capacity is as much as 1μm from the sample surface. Samples for SEM preparations are normally collected by biopsy/ dissection/ sacrificing of an animal to remove tissues from the main body. Incisions should be done with fresh sharp blades to avoid tissue deformation from the physical forces applied with blunt blades. Collected samples should be immediately transferred to vial containing the fixative for fixation. Microbial cultures fixation of bacteria and fungus needs to be carried out under strict safety measures. Biosafety cabinets should be used wherever possible and sample should be removed from the biosafety cabinets only after the

Electron-microscopy, as one of the several tools in diagnostic pathology and cell biology has contributed significantly to reveal several unimagined structures in modern era of science and technology. Several branches of biology and biomedical sciences like anatomy, pathology, physiology, biochemistry, microbiology rely on the transmission electron-microscope. This is used not only to visualize biological materials, but also to analyze the chemical makeup and physical properties of biological materials. Correlate electron microscopic and biochemical studies of cellular processes transcended the boundaries between structurally and functionally oriented sciences. The first electronmicroscope was developed by Max Knoll and Ernst Ruska in 1932 in Germany. Ruska’s efforts were appreciated by awarding him Noble Prize in Physics in 1986. The Siemens Corporation introduced the first commercial transmission electron- microscope in 1938-39 and its commercial marketing started in 1940 by different manufacturers like Hitachi (Japan), JEOL (Japan), Philips (Holland), Zeiss (Germany). Transmission electron-microscope projects electrons through a very thin section of tissue to produce a two dimensional image on a phosphorescent screen. The brightness of a particular area of the image is proportional to the number of electrons that are transmitted through the specimen. A resolution of 0.2 nm can be achieved in the transmission electron-microscope. The first electron-micrograph of biological tissue was published by Marton in 1934. Albert Claude and George Palade were awarded the Nobel Prize (1974) in the field of Medicine for their accomplishments in cell biology employing electron-microscopy

Confocal microscopy is a powerful tool that creates sharp images of a specimen that would otherwise appear blurred when viewed under a conventional microscope. This is achieved by excluding most of the light from the specimen that is not from the microscope’s focal plane.The image thus obtained has less haze and better contrast than that of a conventional microscope and represents a thin cross-section of the specimen. Laser scanning confocal microscopy has become an invaluable tool for imaging thin optical sections in living and fixed specimens ranging in thickness up to 100 micrometers. In fact the confocal microscope is often capable of revealing the presence of single molecule. The modern confocal microscopes can be considered as completely integrated electronic systems, where the optical microscope plays a central role in a configuration that consists of one or more electronic detectors, a computer (for image display, processing, output, and storage) and several laser systems combined with wave length selection devices and a beam scanning assembly. In most advanced systems, integration between the various components is so through that the entire confocal microscope is often collectively referred to as a digital or video imaging, capable of producing electronic images. Principle Current instruments are highly modified from the earliest versions, but the principle of confocal imaging that was developed by Marvin Minsky is employed in all modern confocal microscopes. The image in a confocal microscope is achieved by scanning one or more focused beams of light, usually from a laser or arc-discharge source, across the specimen. This point of illumination is brought to focus in the specimen by the objective lens and laterally scanned using some form of scanning device under computer control. The sequences of points of light from the specimen are detected by a photomultiplier tube (PMT) through a pinhole (or in some cases, a slit) and the output from the

Characterization of Oct-4/Sox-2 Transcription Factors by Immunostaining for Characterization of Stem Cells Protocol 1) The cultured cells at a density of 70,000/well in a 6 well plate or 35,000/ well in a 12 well plate are used for immunostaining. 2) The cells are washed with Dulbecco’s phosphate buffered saline (DPBS) for two times and the cells are fixed using 4% paraformaldehyde for 20 minutes at room temperature. 3) After fixation, the spent Paraformaldehyde is removed and the fixed cells are washed with 1000μl of 1x DPBS for 2 times by clockwise and anti-clockwise rotation of the plate slowly. 4) About 400μl of 3% blocking buffer was added to the wells and plate was covered with aluminum foil to prevent the exposure of light and stored at room temperature for 90 minutes. 5) After blocking, the cells are again washed once with DPBS. About 500 μl of primary antibody Oct-4 in the dilution of 1:40 is added to the well and stored at 4°C over night. 6) After overnight staining with primary antibody, the antibody solution is removed from the wells and washed with DPBS for 2 times. 7) Take 500 μl of secondary antibody anti-rabbit-FITC diluted with antibody buffer solution is added in the ratio of 1:200 and cover with aluminium foil and incubated for 90 minutes. 8) Secondary antibody is removed and washed with DPBS.

Nanotechnology is manipulation of matter on an atomic, molecular, and supramolecular scale. The earliest, widespread description of nanotechnology referred to the particular technological goal of precisely manipulating atoms and molecules for fabrication of macroscale products, also now referred to as molecular nanotechnology. Nanotechnology as the manipulation of matter with at least one dimension sized from 1 to 100 nanometers. This definition reflects the fact that quantum mechanical effects are important at this quantum- realm scale, and so the definition shifted from a particular technological goal to a research category inclusive of all types of research and technologies that deal with the special properties of matter which occur below the given size threshold. It is therefore common to see the plural form "nanotechnologies" as well as "nanoscale technologies" to refer to the broad range of research and applications whose common trait is size. Introduction A nanometer (nm) is one thousand millionth of a meter. A single human hair is about 80,000 nm wide; a red blood cell is approximately 7,000 nm wide, a DNA molecule 2 to 2.5 nm, and a water molecule almost 0.3 nm. The term ”nanotechnology” was created by Norio Taniguchi of Tokyo University in 1974 to describe the precision manufacture of materials with nanometer tolerances, but its origins date back to Richard Feynman’s 1959 talk ”There’s Plenty of Room at the Bottom” in which he proposed the direct manipulation of individual atoms as a more powerful form of synthetic chemistry. Eric Drexler of MIT expanded Taniguchi’s definition and popularised nanotechnology in his 1986 book “Engines of Creation: The Coming Era of Nanotechnology”. On a nanoscale, i.e. from around 100 nm down to the size of atoms (approximately 0.2 nm) the properties of materials can be very different from those on a

Extraction of DNA from Tissue (Using HiPurATM Multi-sample DNA Purification Kit) Kit contents Re-suspension buffer, Lysis solution, Prewash solution concentrate, Wash solution concentrate, Elution buffer, Proteinase K, HiElute Miniprep Spin Column (Capped), Collection tubes (uncapped), Collection tubes Dilute the prewash, wash solution and Proteinase K according to the kit. Protocol Prepare tissue Weigh 25 mg of tissue, add approximately 3 ml of 0.8 per cent of normal saline. Homogenize the tissue with homogenizer or with mortar and pestle. Transfer the homogenized tissue with saline to a microcentrifuge tube. Centrifuge at 14000 rpm for 10 minutes. Discard the supernatant and use the pelleted tissue for further lysis. Digest tissue Add 180 μl of resuspension buffer and 20 μl of Proteinase K solution to the pelleted tissue in microcentrifuge tube. Mix by vortexing. Incubate the sample at 55oC until the tissue is completely digested (2-4 hours) in a shaking water bath. Vortex briefly after complete digestion.

Cellular stains are the colouring compounds used to impart different colours to varied type of cellular components and help in the demonstration of different micro-organisms such as bacteria, viral inclusions, fungal hyphae etc. The staining techniques enhance the contrast of tissue sections at microscopic level including histological, histopathological and cytological grounds. Before applying the special staining techniques on tissue sections few points which should be taken care include: • Use of appropriate volume, concentration and type of fixative for preservation of tissue samples. • Use of fixative with better compatibility as per the nature of tissues to be preserved. • The tissue sections collected should not be very thick (5 mm approximately). • Staining solutions should be immediately prepared where so ever required. • Tissues must be sectioned with an ideal thickness between 4-6 microns. Different types of stains routinely used to demonstrate varied type of cellular components and microorganisms: Grocott’s Methenamine Silver (GMS) Staining Method The fungal hyphae can be better demonstrated with Grocott’s Methenamine Silver stain (GMS). The basis for colouration of fungus is entirely based upon the oxidation of chromic acid forming aldehyde from the cell wall constituents of fungus and thereby reducing the silver-hexamine complex. The silverhexamine complex leads to the reduction of nascent Ag ions and leading the blackening of fungus in tissue sections.

“A Message of Wisdom from Dead to Living” Postmortem examination is systematic exposure and scientific examination of tissue and organs of an animal carcass or cadaver or dead body to determine the nature, extent and cause of death. It is an important diagnostic procedure which supports the diagnosis of a disease in an animal flock or herd. Several times the clinical diagnosis alone may not be sufficient to determine the process of disease occurrence. In such cases gross and histopathological investigations may give a valuable outlook to understand the mechanism of the disease process. Role of necropsy examination 1) Critical necropsy examination complements the clinical medicine by providing a sure short supplement to the clinicians and students. 2) Necropsy examination pave a path for rational diagnosis of cause of death. 3) Necropsy examination helps to assess the line of treatment and diagnostic protocols followed during the illness. 4) Systemic necropsy examination helps to understand the pathogenesis of disease condition in a better manner. 5) Necropsy examination of animals died in a particular region helps to prevent the life of several animals in future. 6) Thorough necropsy examination in animals is an important tool to provide an expert advice to issue Insurance Certificate. 7) Post-mortem examination is often helpful to investigate the death of the animals counted under vetro-legal or forensic aspects.

The constitution of different solutions plays a significant role in successful demonstration of anatomical techniques especially the special stains. The sincerety begins at the time of fixation of the tissues and deviation at any step may lead to failure of procedure. The chemical reagents required for a particular method have been enlisted at their sites however; the commonly used chemicals are comprehended at one place for convenience of the users.