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  • SDS-Polyacrylamide Gel Electrophoresis at Neutral pH (NuPAGE)
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Histochemistry is comprised of two words Histo & Chemistry, which means the chemistry of tissues. In the year 1800, histochemistry became a part of science and now it is one of the most widely used techniques to help scientists localize and visualize cellular components, tissues, and other living structures.[3] This technique uses different stains and indicators, which reacts with the cellular components, to develop tiny colored structures that could be easily observed under a microscope. Histochemistry involves the aspect of both Chemistry and Histology.

Do you know?

In the early phase of Histochemistry, it was limited to looking only into dead cells but with the advancement of research, this field has expanded its horizon to see what’s inside the living tissues or cells! [1]

Techniques/ Methods used in Histochemistry:

There are different techniques used to stain the cells or tissue to observe the colored structures under the microscope. However, cells can’t be directly stained. Before proceeding with the staining part, there are steps[7] involved in coloring the cell;

  1. Animal treatment and tissue processing
  2. Fixation of tissues and cells (chemical fixation and cryo-fixation)
  3. Embedding and sectioning
  4. Staining & observation of specimen by microscopy

Histochemical methods are based on the type of molecule needed to be studied or the structure to be observed. Some of the methods with their principles are explained below.

Perl’s reaction

This technique is based on the principle of the reaction of ferric ions present in the tissues, with ferrocyanide, which results in the formation of Prussian blue color.[2]

In this method, the section of the tissue or specimen is incubated with acid ferrocyanide and then stained with aqueous neutral red or nuclear fast red. Under the microscope, the blue part indicates iron, and background or nuclei is indicated by red and pink color.

Von Kossa technique

This is based on the principle of the reduction of silver nitrate by calcium phosphates, if they are present in the sample tissue or specimen taken.[1]

In this method, the section is dipped in the solution of silver in the light until the calcium (if present) turns black. After this, the section is washed and stained first with a hypo solution and then washed and stained with nuclear fast red. Under the microscope, the black color indicates calcium, red color indicates nuclei, and the cytoplasm is indicated by pink color.

Lipids staining

This involves the use of disparate Lysochromes (Lipid soluble dyes) like Sudan Black B, Nile red, and Oil Red O, etc. The dyes are selected depending on the type of lipid to be studied and observed by light or electron microscopic techniques.[3]

For example:

  • Monarchy method uses Osmium Tetroxide to visualize (by electron microscopy) the fat droplets and triglycerides.
  • Otan method uses Osmium Tetroxide to study the distribution of hydrophilic lipids.
  • Nile blue is used to distinguish (using light microscopy) the distribution of phospholipids and free fatty acids.
  • Oil Red O mainly stains neutral lipids, but at 60֯ C it also stains cholesterol and cholesteryl esters.

Nucleic acid staining

Various methods[7] that are involved in staining of Nucleic acids are as follows:

  • Feulgen’s nuclear reaction: This is the most widely used method which involves the principle of hydrolysis of DNA by HCl which exposes the deoxyriboses. Then Fuchsine reacts with the aldehyde group which colors the DNA in red.
  • S-Bromo-2′ -Deoxyuridine Method: In this method, BrdU is incorporated in DNA and for its visualization, BrdU specific monoclonal antibodies are used. This method is mainly used for the visualization of DNA in cultured cells, smears, and chromosomal spreads.
  • In-situ Hybridization: This method involves the melting of double-stranded nucleic acid and then hybridizing them with DNA or RNA probes having radioactive elements like 125I or 3H or non-radioactive elements like biotin for visualization of nucleic acid.
  • RNA is mainly stained by using basic dyes such as Toluidine Blue and methylene blue.
  • Dyes that are used to stain both DNA and RNA include, Methyl green pyronin stain which is just used to observe the presence of nucleic acid; and Acridine orange which stains DNA in yellow-green and RNA in red-orange color.

Protein staining

Illustration of proteins histochemically is only done when one or more amino acids are very high in their composition in the protein structure. But when the protein to be demonstrated is mixed with other proteins and is in less concentration, their demonstration is done by enzyme activity. Some proteins are identified either by their tissue location or physio-chemical properties.

There are two principles involved in the illustration of protein:

  1. Reaction with amino acids, which after covalent bonding with a dye, highlights the protein.
  2. Reaction of charged dyes with a net charge on the protein (either positive or negative)

Depending upon the type of amino acid to be demonstrated in the protein, there are various methods[3] involved, some of which are explained in the table below:


Amino acid Method Application
Lysine Oxidation by T-Schiff For the demonstration of histones and globin proteins
Arginine Naphthoquinone sulfonate method, Sakaguchi test For the demonstration of granules in eosinophils and histone proteins.
Tryptophan By dimethylaminobenzaldehyde (DMAB) followed by diazonium salt coupling To demonstrate Proenzymes like trypsin and chymotrypsin etc. and also sections of fibrinogen.
Tyrosine Diazotization coupling reaction, Millon’s method ————
Cysteine Ferric-ferricyanide method and mercury orange For the visualization of different proteins.


  • Basic proteins like histones, myoglobin, and ribosomal basic proteins are also stained and localized by staining with Anionic dyes.

Carbohydrates staining

Illustration of carbohydrates is done mainly by the Periodic acid Schiff (PAS) reaction. This reaction is based on the principle of oxidation of carbohydrates in tissues to give aldehyde group, which after reaction with Schiff’s reagent shows carbohydrates of tissue in purple color.[3] Carbohydrates that are stained by this method are Sulphomucins, Proteoglycan, Glycogen, Glycolipids, etc.

The other stains that are used for carbohydrates staining process are alcian blue (which stain sulfomucin and acid glycoproteins), Ruthenium red (to color mucopolysaccharide), and Lectins (for the identification of glycoconjugates, mucin, etc.)

With the advancement of Histochemistry, in 1960, the Enzyme-histochemistry method was introduced in research and biomedical practices.

What is Enzyme-histochemistry?

Enzymes are proteins that catalyze the reactions. They are specifically localized in certain organelles depending upon their function in the particular organelle, like catalase in peroxisome, mitochondria, and chloroplast; succinate dehydrogenase in mitochondria, etc.[6] Due to many pathological conditions, differences in the activity of enzymes and their distribution inside the cells occur which is demonstrated by this technique. This technique is a link between biochemistry, histology, and molecular pathology. It helps in the study of dose-dependent intoxication and the changes in the organism’s pathological condition over time.

Methods to study the enzyme histochemistry depend upon the class of enzyme.

For example:

Hydrolases – Metal Salt Method, Coupling