IHC, ACTH Test

Immunohistochemistry (IHC) involves the process of selectively imaging antigens in cells of a tissue section by exploiting the principle of antibodies binding specifically to antigens in biological tissues. Adrenocorticotropic hormone (ACTH) is a peptide hormone produced by the anterior pituitary gland that stimulates the adrenal cortex. It stimulates the adrenal cortex to secrete glucocorticoid hormones, which help synthesise glucose, catabolism proteins, mobilise free fatty acids and inhibit inflammation in allergic responses. The hormone comprises a long chain of amino acids that share a high degree of homology between humans, cows, pigs and sheep.

It is necessary to know that the Adrenocorticotropic Hormone is the primary Antibody that should be obtained to carry out the test.
The Antigen retrieval device is not needed.
There are two methods of carrying out this test the standard method or enhanced method.
The standard methods:
1.ABC Method-Avidin-Biotin Complex (ABC) Method: ABC method is standard IHC method and one of widely used technique for immunohistochemical staining.
2.LSAB Method-LSAB IHC protocol is based on labeled streptavidin-biotin (LSAB) method. This method utilizes a biotinylated secondary antibody that links primary antibodies to streptavidin-peroxidase conjugate.
The enhanced method:
1.Polymeric Method:
Polymer-based HRP detection systems are widely accepted as the most sensitive in the field of immunohistochemistry.

The uses of applying the methods of Immunohistochemistry includes the following: When the antibodies bind to the antigen in the tissue sample, the enzyme or dye is activated, and the antigen can then be seen under a microscope. Immunohistochemistry is used to help diagnose diseases, such as cancer. It may also be used to help tell the difference between different types of cancer. It can also be used as a predictor for treatment outcome. It can provide with a wealth of information on the expression of specific proteins within the context of tissue structure.

1. Rinse sections in PBS-Triton X-100 (0.025%) for 2×2min
2. Serum Blocking: incubate sections with 3-4 drops of ready-to-use (RTU) normal serum for 30 minutes to block nonspecific binding of immunoglobulin.
3. Primary Antibody: incubate sections with primary antibody at appropriate dilution in antibody dilution buffer for 1-2 hour at room temperature or overnight at 4 °C. Rinse in PBS.
4. Peroxidase Blocking: incubate sections in 3-4 drops of peroxidase blocking solution for 10 minutes at room temperature. Rinse in PBS.
5. Secondary Antibody: incubate sections with 3-4 drops of secondary antibody for 30 minutes at room temperature.
6. Rinse in PBS for 3×2min.
7. Detection: incubate sections with 3-4 drops of streptavidin-HRP for 30 minutes at room temperature.
8. Rinse in PBS for 3×2min.
9. Chromogen/Substrate: incubate sections with 3 drops of DAB solution for 2-8 minutes. Monitor signal development under a microscope
10. Rinse in PBS 2×2min.
11. Rinse in distilled water 2×2 min.
12. Counterstain: For using hematoxylin counterstain, incubate sections with 3 drops of hematoxylin for 1-2 minutes. Rinse in distilled water. Incubate sections with 3 drops of bluing solution for 1 minute.
13. Rinse in distilled water.
14. Dehydrate through 75% ethanol for 2 min, 95% ethanol for 2 min, and 100% ethanol for 2×3min. Clear in xylene for 2×5min.
15. Coverslip with mounting medium.