Histochemistry is an important technique that is used for the visualization of biological structures. As such, it is concerned with the identification and distribution of various chemical components of tissues through the use of stains, indicators as well as microscopy.
Essentially, identification and distribution of chemical constituents of tissues is achieved through the exploitation of unique chemical environments in cells, heterologous expression techniques as well as enzymatic activities.
This method is particularly important for the detection of ion levels (ferric ions). Because it can help detect the presence of ferric ions, this technique is used to determine the level of these ions in such organs as the spleen and bone marrow. It can be used to tell whether there are excessive amounts of the ion as observed in hemochromatosis (excessive levels of ferric ions with deposits in the liver and pancreas) or hemosiderosis where deposits can be found in the liver, spleen and the lymph nodes.
In plants, this technique has also been shown to help understand the homeostasis of ferric ions.
With this technique, ferric ions present in the tissue will combine with ferrocyanide resulting in the formation of a pigment called Prussian blue (ferric ferrocyanide). In plants, the technique is also based on the conversion of ferrocyanide into insoluble crystals (Prussian blue) in the presence of Ferric ions under acidic conditions. Prussian blue (resulting from the reaction) is bright blue in color, which indicates the presence of ferric ions.
In Perl's reaction technique, a known positive control tissue is used as control while 10 percent formalin is used as a fixative.
* It's important to use gloves, goggles and a lab coat because some of the chemicals used for this procedure can cause irritation.
When viewed under the microscope, blue parts are indicative of iron while the red and pink parts indicate the nuclei and background respectively.
This is also an ion based technique that is used in histochemistry. It is a more sensitive technique that can be used to identify the presence of calcium deposits on cyst fluids, ductal ectasia and papillomatosis. However, excessive amount of calcium may be found in any given part of the body and can be demonstrated using the Von Kossa technique.
Although this technique is used to demonstrate the presence of calcium, it demonstrates an anion rather than the calcium ion itself.
For this technique, the sample section is treated with the solution of silver nitrate and is reduced and the calcium (if present in the sample) is reduced by the strong light and replaced with deposits of silver. As a result, it's visualized as metallic silver.
Requirements for Von Kossa technique
A black color indicates the present of calcium (calcium salts), red indicates the nuclei while the cytoplasm will appear pink.
This technique is dependent on dyes that are soluble in lipids.
Some of the most common dyes used include:
Lipid staining is a useful technique that is used for demonstrating intracellular lipids in various tissue sections.
For this technique, the dye is more soluble in the lipid, which allows it to be more demonstrated than in the vehicular solvent. The dyes used in this technique are all interchangeable, which means that they can be substituted for each other for the staining process.
Red color indicates the lipid while blue coloration indicates the nuclei.
Lipid staining technique is useful for showing the normal distribution of lipids as well as disease-related lipid accumulation.
Some of the methods used for specific amino acids include:
Millon's reagent is used for detecting amino acid tyrosine.
In this technique, the mercurous and mercuric nitrate (components of the reagent) reacts with hydroxybenzene radicals to form a compound that is red in color. Tyrosine contains the phenolic group, which forms the red coloration in the presence of Millon's reagent. The compound formed through this reaction is called mercuric fumarate.
Milton's method procedure:
If the solution turns reddish in color after boiling, then tyrosine is present in the solution.
The Sakaguchi reaction test involves the use of the Sakaguchi reagent. This reagent is composed of 1-naphthol and sodium hypobromite and forms a reddish compound when mixed with the sample containing a Arginine.
The test is positive for any amino acid that contains the guanidine group in Arginine. Therefore, the Guanidine group in an amino acid will react with the α-Naphthol and alkaline hypobromite in the reagent to give of a red-colored complex indicating the presence of such amino-acids.
Sakaguchi's test procedure:
Although it has been shown to be less effective, Tetrazotized benzidine is used in histochemistry to detect non-collagen proteins. The procedure involves the coupling of Tetrazotized benzidine with beta naphthol or Hyaluronic acid.
The method, commonly referred to as Tetrazotized benzidine helps in the detection of such non-collagen proteins as
tyrosine, histidine as well as tryptophan.
This is a relatively new technique that is used for demonstrating DNA in tissue sections. It is a sensitive means of detecting aldehydes, which makes it the ideal method for detecting the presence of DNA. Here, the section is treated with dilute hydrochloric acid in order to remove the bases.
The sugar part that remains reacts as an aldehyde ultimately forming a visible color.
Therefore, this method can be said to be divided in to two main parts:
1/ The first part of the procedure is the hydrolysis phase that involves the use of 5N HCl, ambient temperature for 40 minutes. This step is aimed at separately selecting 2 purine bases (adenine and guanine) which are removed from the DNA molecule.
2/ The second step is the staining phase. The reagent used is preferred because it is highly selective for DNA rather than RNA. Here, RNA does not react because of the presence of hydroxyl on carbon 2 of ribose, which prevents the acid (HCl) from hydrolyzing sugar. The reaction is also precise for the localization of DNA given that deoxyribose radicals are bound to phosphoric acid of the apurinic acid molecule following the removal of purine bases.
Some of the stains used for both DNA and RNA include:
This is one of the most popular histochemical techniques for the detection of glycogen. It has been shown to be one of the best techniques for demonstrating carbohydrates in tissue.
In this technique, the periodic acid oxidizes tissue carbohydrates to produce aldehyde groups. This group then condenses with the reagent to form a bright red coloration to demonstrate the tissue component with carbohydrate attachments.
The diastase and a-amylase in the reagent act on the glycogen and depolymerize it into smaller sugar units (maltose and glucose) which are then washed out of the section.
Some of the other stains used for staining sacchrides include:
Whereas histochemistry includes a number of techniques used for the visualization of various chemical components in tissues, immunochemistry involves the study of identities and functions of components of the immune system (particularly antibodies).
Essentially, immunochemical methods are based on the selective, reversible and non-covalent binding of antigens by antibodies. This allows for these methods to be used to quantify antigens or antibodies.
All the immunochemical methods/techniques depend on a highly specific and sensitive reaction between antigens and antibodies. There are a number of immunochemistry techniques based on the type of reaction, reagents and samples that are used.
Particle methods - This is the technique where the antigen-antibody interaction is observed. It includes a number of methods such as Immunoprecipitation, Immunoelectrophoresis, Immunofixation.
Label methods - With label methods, either the antigen or the antibody is labeled allowing for the antigen-antibody reaction to be observed. Immunoassay and competitive binding are examples of label methods.
Some of the other methods include:
Relevant: How does a Microtome work?
Boguslaw Samotus, Maria Leja, Andrzej Scigalski, Jerzy Dulinski, Robert Siwanowicz (1982) Determination of tyrosine by a modified Millon's reaction and its application to potato tuber extracts.
Culling C.F.A., (1974) Handbook of histopathological and histochemical techniques Ed. 3
Butterworth, London, UK.
Dandekar (1 January 2004). Practicals And Viva In Medical Biochemistry. Elsevier India. p. 28. ISBN 978-81-8147-025-6.
Kiernan. J.A., (1999) Histological and histochemical methods: Theory and practice, Ed. 3
Butterworth Heinemann, Oxford, UK.
Luna, Lee G (1960) Manual of Histologic Staining Methods of the Armed Forces Institute of Pathology (Third Edition). American Registry of Pathology.
Symonds DA (1990) Use of the von Kossa stain in identifying occult calcifications in breast biopsies.
Suzuki M, Shinohara Y, Fujimoto T (2013) Histochemical detection of lipid droplets in cultured cells.