eChapters

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

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

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).

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.

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.

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:

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.

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.

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

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.

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

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).

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.

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.

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.

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

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.

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.

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.

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