detecting chemical components of tissues

1. BELGOROD STATE NATIONAL
RESEARCH UNIVERSITY
MEDICAL INSTITUTE
DEPARTMENT OF HISTOLOGY
TOPIC - HISTOCHEMISTRY
BELGOROD-2016
PRESENTED BY
SOURABH TIWARY
GROUP - 03011424
2nd course

2. Planning of report
1. What is histochemistry ?
2. Goals of histochemistry
3. About histochemistry
4. Terms and special disciplines within
histochemistry
5. Methods for detecting biochemical
substances
6. Applications of histochemistry
7. Limitations of current method
8. Reference

3. What is histochemistry ?
 It is the branch of science concerned with the
identification and distribution of the chemical
constituents of tissues by means of stains,
indicators, and microscopy.

4. Goals of histochemistry
 Presntation of the normal chemical composition
 Presentation of the normal chemical distribution
 Specificity of reaction
 Detectibility of reaction product
 Insolubility of reaction product

5. Related terms and special disciplines
within histochemistry:
1. Cytochemistry
2. Enzyme histochemistry
3. Immunocytochemistry
4. In situ hybridization

6.  Cytochemistry is the biochemistry of cells, especially that of the
macromolecules responsible for cell structure and function. The
term is also used to describe a process of identification of the
biochemical content of cells.
 Enzyme histochemistry serves as a link between biochemistry and
morphology. It is based on metabolization of a substrate provided
to a tissue enzyme in its orthotopic localization. Visualization is
accomplished with an insoluble dye product. It is a sensitive
dynamic technique that mirrors even early metabolic imbalance of
a pathological tissue lesion, combined with the advantage of
histotopographic enzyme localization.

7.  Immunocytochemistry (ICC) is a common laboratory technique that is
used to anatomically visualize the localization of a specific protein or
antigen in cells by use of a specific primary antibody that binds to it.
 In situ hybridization (ISH) is a type of hybridization that uses a labeled
complementary DNA, RNA or modified nucleic acids strand (i.e.,
probe) to localize a specific DNA or RNA sequence in a portion or
section of tissue (in situ), or, if the tissue is small enough (e.g., plant
seeds, Drosophila embryos), in the entire tissue (whole mount ISH), in
cells, and in circulating tumor cells (CTCs). This is distinct from
immunohistochemistry, which usually localizes proteins in tissue
sections.

8. Some important biologic
substances & classic
methods for detecting
them:

9. Examples of Histochemical Methods:
1. Ions
Iron (ferric ions – Fe3+)
• Perls’s reaction: – sections of tissues are incubated in a
mixture of potassium ferrocyanide and hydrochloric acid
– result: insoluble dark blue precipitate of ferric
ferrocyanide
• Diagnostic application: – patients with diseases that
store iron (eg. hemochromatosis)

10. Perlis reaction –detection of Fe3+
Fe3+ (HCl) and potassium ferrocyanide.
Product of reaction is Prussian blue

11. Calcium Phosphate: Von Kossa technique
Tissue phosphates react with silver nitrate to form silver
phosphate, which reacts with hydroquinone to form a
black precipitate of reduced silver.
This reaction is used to study calcium phosphate
deposition during bone formation

12. Von kossa technique

13. 2.lipids
 stained with dyes soluble in the lipids
 eg. Sudan IV, Sudan black, Oil red 0, Nile
blue for light microscopy.
 For EM , osmium tetroxide.
 Such methods are used to show normal lipid
distribution and disease-related lipid
accumulation (eg, fatty change in the liver).

14. Steatosis of the liver (fatty liver) seen as
red globules in the Oil Red O stain

15. 3. Proteins and Amino Acids
Older methods of protein identification are nonspecific for proteins
but specific for particular amino acids.
Examples: Million reaction for tyrosine, Sakaguchi reaction for
arginine, tetrazotized benzidine reaction for tryptophan.
 Specific classes of enzymes can be detected by the techniques of
enzyme histochemistry.
 Specific proteins can now be localized by using immuno-
histochemistry.

16. 4.Nucleic Acids – DNA
 Feulgen’s reaction:
– hydrolysis of DNA by hydrochloric acid
– this process leads to the formation of
aldehyde groups
– free aldehyde groups react with the Schiff
reagent
– result: insoluble red substance

17. Fuelgen reaction- acinar cells staining

18. 4.Nucleic Acids – RNA
• RNA-rich organelles are stained with basic dyes
• i.e.: toluidine blue, methylene blue
 In this procedure, one of 2 adjacent sections is
treated with ribonuclease (RNase) to remove
RNA; then both are stained with basic dyes (eg,
hematoxylin, toluidine blue, methylene blue).

19. Some common staining for both nucleic
acids
 Methyl Green Pyronin Stain to determine DNA and
RNA
 Acridine orange: The fluorescence is yellow green if the
complex contains DNA and red-orange if it contains
RNA.

20. 5.Saccharides
• Saccharides can be detect by PAS reaction
(periodic acid-Schiff)
• it is based on oxidative action of periodic acid
(HIO4) → aldehyde groups
• these aldehyde groups react with Schiff’s reagent
(as in Feulgen’s reaction)
• → a new compound with a purple colour (PAS-
positive substances).
 Lectins ,Ruthenium red, Alcian blue are also used for
staining of sacchrides.

21. Saccharides cont….
• PAS-positive substances are:
– polysaccharides (glycogen)
– glycosaminoglycans /mucopolysaccharides/ (hyaluronic
acid, chondroitin sulphate)
-proteoglycans
– glycoproteins (thyreoglobulin, collagen)
– glycolipids (lipofuscin)
• Clinical application: – biopsies of tissues from patients with
diseases that store glycogen (glycogenosis),
glycosaminoglycans (mucopolysaccharidosis)...

22. Glycogen staining by PAS reaction

23. Enzyme Histochemistry
• Enzymes are the catalysts of most of biochemical reactions
• The techniques of enzyme histochemistry, which relate structure
and function, can be used to locate many enzymes, including acid
phosphatase, dehydrogenases, and peroxidases.
Because fixation and clearing typically inactivate enzymes, frozen
sections are commonly used.
• Principle:
– 1st reaction (histochemical):
enzyme + substrate → product
– 2nd reaction:
demonstration of the product

24. Enzyme Histochemistry – Examples
• Peroxidase
– sections are incubated in a solution containing hydrogen
peroxide and DAB (diaminoazobenzidine)
– DAB is oxidized in the presence of peroxidase
– result: insoluble, black, electrondense precipitate
• Clinical application:
– diagnosis of leukemias (detection of the peroxidase activity
in blood cells)
• Peroxidase is used also as a label in immunocytochemistry
and in ISH

25. Immunocytochemistry
 Is a technique for identifying cellular or tissue constituents (antigens) by
means of antigen-antibody interactions
 Antigens:
– proteins, glycoproteins, proteoglycans
 Antibodies:
– serum proteins known as immunoglobulins
– formed in the humoral immune system by plasma cells
– there are five types of antibody found in the blood IgA, IgD, IgE, IgG and
IgM.
– IgG is the commonest and the most frequently used antibody for
immunohistochemistry

26. Methods of Labelling Antibodies
• Enzyme labels (peroxidase)
• Colloidal metal labels (EM)
• Fluorescent labels
• Avidin-biotin techniques

27. References -
 Classroom lek.
 Wikipedia
 Linked in .com
 Slideshares.com

28. THANK YOU FOR
YOUR GRAND
ATTENTION