A TERM PAPER ON

HISTOCHEMICAL TECHNIQUES OF PROTEINS

PRESENTED BY GROUP 3

ERIN JADESOLA (BU20ANA1018)

ESUOLA OLUWAFEMI (BU20MED1047)
AJETUNMOBI DEBORAH (BU20MED1015)
ABIOLA OYINDOLAPO (BU20ANA1024)

ANA 403- HISTOCHEMISTRY

Table of contents

1. Introduction to proteins

2. Types and functions of proteins

3. Properties of proteins

4. Tissue preparation

5. Histochemical techniques for protein demonstration

6. Applications in pathological conditions

7. Conclusion

8. References
INTRODUCTION TO PROTEINS

Proteins are large molecules that are involved in all cell functions (Brody, 2020). It is made up of

amino acids, about 20 common acids. The structure of an amino acid constitutes a central carbon,

an amino group (-NH2) and a carboxyl group (-COOH). Proteins are found in the muscles, liver,

hair, bones and other organs and tissues in the body (Koshland &Haurowitz, 2023). Amino acids

can be divided into essential and non- essential. Essential amino acids cannot be synthesized by

the body. Hence, they have to be consumed in diet. Essential amino acids include Histidine,

Isoleucine, Leucine, Lysine, Methionine, Phenylalanine, Threonine, Tryptophan and Valine.

The non-essential amino acids are synthesized by the body. They include: Alanine, Arginine,

Asparagine, Aspartic acid, Cysteine, Glutamic acid, Glutamine, Glycine, Proline, Serine and

Tyrosine (Smith, 2022)

TYPES AND FUNCTIONS OF PROTEINS

Enzymes, catalysts in reactions, are proteins. These enzymes could be either catabolic (breaking

down of substrates) or anabolic (building up of complex molecules from substrates). Hormones,

secreted by the endocrine cells are usually either proteins or steroids (Bailey, 2020; Chruścik et

al., 2021). The following are types and functions of proteins with their examples (Chruścik et al.,

2021):

1. Enzymes: They help in breaking down nutrients into monomeric units. Salivary amylase

in saliva helps to breakdown starch to sugar. Pepsin also helps in the breakdown of

proteins to peptides.

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2. Transport: These ones carry substances in the blood or lymph throughout the body.

Haemoglobin transports oxygen through blood via red blood cells.

3. Structural Protein: They form different structures. Examples are collagen (found in the

bone and skin), actin, keratin (strengthens skin and hair).

4. Hormones: They coordinate the activities of the systems of the body. Insulin in the body

regulates glucose metabolism while oxytocin stimulates contractions during childbirth.

5. Defence Proteins/Antibodies: They protect the body from foreign pathogens e.g.

Immunoglobulins.

6. Contractile Proteins: They aid muscle contraction. They are actin and myosin.

7. Storage: These store amino acids until the body is ready to use them. Ferritin stores iron

in haemoglobin, for instance.

PROPERTIES OF PROTEINS (MN Editors, 2023)

1. Solubility: Proteins usually form colloidal solutions rather than pure solutions in

water.

2. Molecular weight: The molecular weight of proteins vary depending on how many

amino acids residues a protein contains. For instance, serum albumin weighs

approximately 69,000 molecules while insulin has a molecular weight of 5,700.

3. Shape: Proteins come in a variety of shapes, from insulin’s globular forms to

fibrinogen’s elongated structures.

4. Isoelectric PH: The amino acid composition of each proteins determines its isoelectric

PH (pI).

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5. Acidic and Basic Proteins: A protein’s nature is determined by the proportion of basic

to acidic amino acids in it. Proteins classified as basic are those whose ratio is greater

than 1 and those classified as acidic are those whole ratio is less than 1.

TISSUE PREPARATION

Before the demonstration process of protein using histochemical methods, general

protocols for histochemical study have to be followed. These protocols according to Rolls

(2021) include:

1. Obtaining a fresh specimen: It is important that fresh tissues are handled with care.

Fixation should take place at the site of removal and if this is not possible, fixation

should happen right after the sample is transported to the lab.

2. Fixation: A liquid fixing agent, usually formaldehyde solution (formalin) is used.

Fixatives help to harden and preserve tissue against subsequent tissue preparation

steps. The specimens should be left in the fixative for sufficient amount of time to

allow the fixative permeate areas of the tissue and for an additional amount of time to

allow the equilibrium of the chemical reactions involved in fixation. The specimen

should fix for between 6 and 24 hours.

3. Dehydration: Infiltration is done by paraffin wax. And so, because paraffin wax is

immiscible with water, the majority of water must be eliminated before the wax can

penetrate. To do this, the specimen is submerged in a succession of ethanol (alcohol)

solutions of increasing concentration. To prevent undue tissue distortion, a sequence

of concentration increase is employed. A typical dehydration sequence for specimens

no thicker than 4mm might be:

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 70% ethanol for 15 minutes

90% ethanol for 15 minutes

100% ethanol for 15 minutes

100% ethanol for 30 minutes

100% ethanol for 45 minutes

At this point, the specimen should have a minute amount of water left.

4. Clearing: Even when the tissue is water-free, it still cannot be treated with wax yet as

wax and ethanol are immiscible too. As a result, an intermediate solvent that is

completely miscible with paraffin wax and ethanol must be utilized. The reagent used

is known as a clearing agent and this procedure is known as clearing. Because of their

high refractive index, many but not all clearing agents, give the tissue a form of

transparency. Another function of the clearing agent is to remove excess fat in the

tissue which may serve as a barrier to wax infiltration. Xylene is a common clearing

agent.

5. Wax Infiltration: A suitable histological wax can now be used. Paraffin wax is the

most popular. At 60℃, a common wax is liquid and can be injected into tissue. Once

the temperature drops to 20℃, the wax freezes into a consistency that makes cutting

sections of tissue reliably possible.

6. Embedding: The specimen needs to be shaped into a block like form that can be

clamped into a microtome for section cutting after infiltration with wax. This

procedure is done with an embedding center where the specimen is inserted into a

mold that has been filled with molten wax. After adding more wax to a cassette that

sits on top of the mold, the entire assembly is set aside to solidify on a cold plate.

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 Upon completion, the block can be taken out of the mold along with the cassette it is

attached to, making it prepared for microtomy.

7. Sectioning: According to an atlas of comparative vertebrate histology (2018), a

microtome, a specialized precision cutting tool is used to repeatedly and precisely

slice sections from an embedded tissue block. A sharp microtome and the tissue block

are held in a fixed relationship in every microtome. The tissue block advances by a

predetermined amount- the section thickness, with each pass of the tissue past the

knife. The typical thickness of a section is 8 to 15μm for frozen sections and 4 to

10μm for wax sections.

A pathological laboratory technique called frozen section (cryosection) is used to

quickly diagnose a disease or perform analysis of a specimen. It is used in

conjunction with oncologic surgery (Wallace, 2023). A cryostat is the microtome

used to cut frozen tissue.

HISTOCHEMICAL TECHNIQUES FOR PROTEIN DEMONSTRATION

According to ConductScience (2019), the histochemical techniques for proteins is based

on their tissue location and their amino acid composition.

The amino acids needed for this demonstrations (such as tyrosine, cysteine) must be

present in very high concentration at the tissue location in order for the demonstrations to

be carried out. However, they are typically found in low concentrations, mingled with a

variety of other proteins, which makes their demonstration more difficult. Here, the

biological characteristics of the protein, such as enzyme activity are used to demonstrate

it.

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Some staining techniques for fibrous protein demonstration include:

1. Van Gieson stain (Ellis, 2023; Srinivas, 2016): This is used to show how collagen

increases and to distinguish between collagen and smooth muscle in tumours. It is a

mixture of picric acid and acid fuschin. When picric acid and acid fuschin are

combined, the picric acid’s small molecules quickly permeate all tissues, but they are

firmly retained in muscle and red blood cells.

Formulas for reagents:

- Celestin blue – 100ml of 5% ammonium ferric sulphate- 0.5g.

- 90 ml of saturated aqueous picric acid, 10 ml of 1% acid fuschin.

Method

- Celestin blue or Weigert’s iron hematoxylin is used to stain the nuclei.

- Rinsing in water

- Staining and differentiation of nuclear stain in Van Gieson’s solution for 1-5

minutes.

- Dehydration in absolute alcohol.

- Clearing in xylene and mounting.

Result: Collagen-red

Muscle tissue- deep yellow

Nuclei-brown

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 Van Gieson trichome stain showing the deep region of the dermis

©Megias M, Molist P, Pombal MA (2019). Atlas of plant and animal histology

2. Masson Trichome Stain (Srinivas, 2016; Mokobi, 2020): This staining consists of

three stains and is used for the detecting collagen fibers in tissues such as the skin,

heart and muscle. Reagents:

Solution A- 0.5g of acid fuschin, 0.5ml of glacial acetic acid, 100ml of distilled

water.

Solution B- Phosphomolybdic acid-1g, distilled water- 100ml

Solution C- Methyl blue-2g, Glacial acetic acid- 2.5ml, distilled water- 100ml.

Bouin fluid- Saturated picric acid-75ml, 40% formaldehyde-25ml, and glacial acetic

acid-5 ml.

Method:

- Sections are deparaffinised using 100% alcohol, 95 % alcohol and 70% alcohol

and are place under water.

- Tissues that have been fixed with formalin are re-fix in Bouin Solution for an

hour at 56℃.

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- Sections are washed under tap water.

- Nuclei is stained by Celestine-blue hematoxlyin method.

- Differentiation by 1% acid alcohol.

- Wash under tap water

- Stain in Acid Fuschin solution A for 5 minutes

- Rinse in distilled water then treat phosphomolybdic acid solution B for 5 minutes

- Stain with methyl blue solution C for 2-5 minutes

- Rinse with distilled water and treat with 1% acid for 2 minutes

- Dehydrate through ascending grades of alcohol

- Clear in xylene and mount on permanent mounting medium.

Results: Nuclei- blue/black

Cytoplasm, muscle, keratin and RBCs- Red

Collagen- blue

Masson’s trichome staining of collagen within the liver tissue

© Hend Maarof Tag, 2015.

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For other proteins:

1. Sakaguchi test (Arginine reaction): Using this technique, the test sample’s

arginine content is determined.

Principle: α naphthol and a single drop of sodium hypochlorite, sometimes

known as Sakaguchi reagent, makes up the reagent used in this procedure. After α

naphtol and arginine’s guanidyl group react, sodium hypochlorite oxidizes the

resulting product giving a red colour.

Materials: α naphthol, pipettes, dry test tubes, sodium hypochlorite and urea.

Method: A test tube is filled with 5ml of the test sample. 1ml of 10% NaOH and

1 ml of α naphthol are added as well as thoroughly mixed. 1 ml of sodium

hypochlorite is added to the tube. 2ml of urea of urea solution is added after the

sodium hypochlorite. The tube is then thoroughly stirred.

After 2 to 3 minutes, a reddish-coloured solution might be observed indicating

that sample contains arginine.

2. Hopkin’s Cole Test (Arginine reaction): The tryptophan content of a sample is

determined using this technique. Proenzymes, gonadotropins and glucagon all

contain high concentrations of tryptophan.

Principle: The purple colour of the solution is caused by the reaction between the

sample’s tryptophan and glyoxylic acid in the presence of conc. H2SO4.

Requirements: Dry test tubes, pipettes, conc. H 2SO4, test sample, Adam Kevich;s

reagent (made by reducing oxalic acid with magnesium powder or sodium

amalgam).

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 Method: The test tube is filled with 2 ml of the test sample. 1 ml of Adam

Kevich’s reagent is added to the test tube and mixed thoroughly. 1 ml of conc.

H2SO4 is added carefully along the side of the test tube. Tryptophan is present in

the sample when a purple ring forms at the junction of the two solutions in 2 to

three minutes.

3. Millon’s Test (Tyrosine reaction): Tyrosine’s presence in the test sample is

determined using this method.

Principle: Nitric acid nitrates the phenol group of tyrosine. After that, this

product combines with mercury to form a complex, giving the solution a pink to

brick red hue.

Materials: Test tubes, Millon’s reagent (a mercury nitrate-nitric acid solution),

and the test sample are used.

Method: 1 or 2ml of the test sample are placed in a test tube. The test sample is

filled with 1 ml of Millon’s reagent and thoroughly mixed. The solution is then

heated until it boils if there is no noticeable change in colour. Sample does not

contain tyrosine if there is no observable colour change.

4. Ninhydrin Test: Proteins, peptides and amino acids can all be found in the test

sample using this method. Proteins and amino acids that have a α-amino group

interact with ninhydrin.

Principle: Ninhydrin is an extremely potent oxidizing agent. An amino acid

undergoes oxidative deamination upon reaction with an amino group which results in

the reduced forms of ammonia, CO 2 and ninhydrin. The reduced ninhydrin forms a

blue-coloured complex by reacting with other ninhydrin and released ammonia.

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 The following are needed: test sample, phenol, acetate buffer, pipette and ninhydrin

reagent (0.6g of ninhydrin dissolved in 10ml of absolute ethanol).

Method: In a test tube, 1 ml of the tests sample is taken. The test sample tube is

filled with 5ml of phenol and 0.5ml of buffer. To obtain a transparent solution, the

mixture is given a good shake. O.5ml of ninhydrin is added and thoroughly mixed

with the other constituents of the test tube. The test tube is then submerged in water

for about 5 minutes. Water is used to cool it. A purple coloured solution indicates that

protein is present. A light yellow colour indicates that the test is negative.

Note: Proline will display a yellow colour solution because it lacks a free α-amino

group.

Other staining methods:

These other staining methods are done after electrophoresis (separation of proteins based on size

and electrical charge) has been done. They include:

1. Silver stains (Conductscience, 2019): This is used because silver binds to the terminal

end or side chain of an amino acid. In the silver staining process, fixation occurs first in

which the protein molecules are immobilized and interfering substances are eliminated.

After that, substances are added to the gel to either speed up the reduction of silver or

cause proteins to become reactive to it. Following that, either ammoniacal silver or plain

silver nitrate are used for silver impregnation. After rinsing the gel, the silver metal image

is finally obtained. The amount of silver bonded to the protein bands determines the

different shades that are produced in the gel.

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 2. Zinc Stains: This is one popular staining technique for identifying proteins fractionated

by polyacrylamide gels (Conductscience, 2019). Because the process is reversible, there

is less chance of protein modification. The basis of the reversible zinc staining method is

the way that zinc ions (Zn2+) interact with proteins and biomolecules, Because of the

sodium dodecyl sulphate coating on the proteins, the stain is unable to bind to the

proteins and instead precipitates zinc metal in the gel turning it opaque white. This

creates a negative image that makes it possible to see distinct protein bands on top of a

semi opaque white polyacrylamide background (Conductscience, 2019).

3. Coomassie Brilliant Blue (Excedr, 2022): The Coomassie Brilliant Blue dye family is

widely utilized in laboratory settings for protein staining procedures which are carried out

after polyacrylamide gel electrophoresis. These protein dyes’ staining chemistry is

determined by how well they bind to protein and matrix. The basic amino acid residue of

the proteins and the dye combine to form a complex. There are 2 types of Coomassie

brilliant blue- Coomassie brilliant blue G-250 and Coomassie brilliant blue R-250.

APPLICATIONS OF HISTOCHEMICAL METHODS OF PROTEIN

DEMONSTRATION IN PATHOLOGICAL CONDITIONS (Conductscience, 2019)

1. Neurosecretory material: There is more cysteine in the posterior pituitary

hormones and their carrier protein than in any other protein or peptide.

Cysteine oxidation and reduction techniques are widely employed in the

investigation of these hormones and their carrier proteins.

2. Crooke Russell Cell: Muco-protein rich in cysteine are found in certain

pituitary basophils, such as the Crooke- Russell cell. This technique can be

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 used to observe basophils, analyze ACTH (Adrenocorticotrophic Hormone)

and research Addison’s diseases.

3. Melanocyte stimulating hormone: This hormone causes melanocytes to be

secreted more frequently, giving the skin a darker appearance. Tryptophan is

abundant in this hormone, so demonstrating tryptophan in this condition can

be used in determining the hormone’s level.

4. Serotonin (5-hydroxytryptamine): This hormone is a tryptophan derivative.

The tryptophan demonstrating method in conjunction with the carboline

method can be used to study the pathological condition related to argentaffin

granules (granules rich in serotonin, which helps in digestion) and cancer

(serotonin acts as a growth factor in many carcinomas and gliomas).

5. Neurokeratin: When this material was initially isolated from the brain, it was

discovered to be protease-resistant. Tryptophan is found in greater quantities

in neurokeratin than in any other amino acid and it is abundant in myelin

sheath. Thus, tryptophan demonstrating histochemical methods can be used to

study any disorder related to the myelin sheath.

6. Protein malnutrition: The body is impacted by high or low protein intake in

many different ways e.g. kwashiorkor disease. Techniques that show

tryptophan (in muscle) and cysteine, tyrosine and arginine (in the epidermis)

can be used in this study.

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 CONCLUSION

Proteins are involved in every-day life processes. Generally, histochemistry

allows us to visualize and study the distribution, location and expression

levels of proteins within tissues. It provides information about the presence

and abundance of proteins in different cell types. Histochemical methods for

protein demonstration have contributed largely to research study of different

biological structures and pathological conditions.

REFRENCES

An atlas of comparative vertebrate histology. (2018). Microtome - an overview | ScienceDirect

Topics. Www.sciencedirect.com.

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Bailey, R. (2020, January 23). The Complex Nature of Proteins in Cells. ThoughtCo.

https://www.thoughtco.com/protein-function-373550

Brody, L. (2020). Protein. Genome.gov. https://www.genome.gov/genetics-glossary/Protein

Chruścik, A., Kauter, K., Windus, L., & Whiteside, E. (2021). 2.4 Protein. Usq.pressbooks.pub.

https://usq.pressbooks.pub/anatomy/chapter/2-4-protein/

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Conductscience. (2019, June 30). Silver Staining Protocol. Conduct Science.

https://conductscience.com/silver-staining-protocol/

Ellis, R. (n.d.). van Gieson Staining Protocol. Www.ihcworld.com. Retrieved December 6, 2023,

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 from https://www.ihcworld.com/_protocols/special_stains/van_gieson_ellis.htm

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https://www.excedr.com/resources/coomassie-blue-staining

Koshland, D. E., & Haurowitz, F. (2023, November 11). Protein - General structure and

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%20frozen%20section%20(cryosection)%20is

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