Immunohistochemistry or IHC refers to the process of detecting antigens in cells of a tissue section via antigen retrieval. This is carried out by exploiting the principle of antibodies binding specifically to antigens in biological tissues.
Immunohistochemical staining is widely used in the diagnosis of cancerous tumors from hodgkins lymphoma to differentiation of different tumours eg ductal carcinoma in the breast based on specific antigen staining. Specific molecular markers are characteristic of particular cellular events such as proliferation or cell death apoptosis. IHC is also widely used in basic research to understand the distribution and localization of biomarkers and differentially expressed proteins in different parts of a biological tissue. Visualising an antibody-antigen interaction can be accomplished in a number of ways. In the most common instance, an antibody is conjugated to an enzyme, such as peroxidase, that can catalyse and make the area visible.
The discipline of Immunohistochemistry or Immunocytochemistry was known since the 1930s but it was really made possible only in 1942 when the first IHC study was reported by Coons. Here he used FITC-labeled antibodies to identify antigens in infected tissue. Since then, improvements have been made in protein conjugation, tissue fixation methods, detection labels and microscopy, making immunohistochemistry a routine and essential tool in diagnostic and research laboratories.
To carry out the procedure the general protocl used below are followed, Sections cut at 3mm and floated onto superfrost glass slides. Sections are drained and then left in the 56C incubator for one hour or at room temp overnight.
1 Deparaffinise Paraffin sections in Xylene – 5 mins. (for Resin sections, De-resin in Xylene overnight)
2 Rinse through graded alcohols to water – 5 mins in each.
3 Wash in water - 5 mins
4 Block with 3% Aqueous Hydrogen peroxide - 10 mins
5 Wash in water - 5 mins
6 Apply antigen Retrieval as applicable:
a) Microwave at full power in CC1 – 2 x 10 mins Remember to top up fluid level after the first 10 minutes
b) Or apply Pronase 1 reagent for the appropriate time in the humidity chamber
7 Wash in running cold water and allow to cool – 5mins
8 Wash in Reaction Buffer and apply Dako Pen around sections
9 Place sections into the humidity box
10 Apply sufficient prediluted Primary antibody to cover the sections for the appropriate time.
11 Rinse with Reaction Buffer – 2 x 5min
12 Apply the Polymer Link (Universal HRP Multimer)- 30 mins
13 Rinse with Reaction Buffer – 2 x 5min
14 Apply the Universal DAB Chromogen, (2 – 3 drops)
15 Add the same amount of Universal DAB H2O2, (2 – 3 drops) to activate the chromogen
16 Visualise microscopically to determine optimal staining. Then wash well in water.
18 Counterstain lightly in haematoxylin for about 15 seconds, then Wash well in water to “blue-up” sections
19 Enhance staining by placing slides in 0.5% Aqueous CuSO4 for 10 minutes
20 Wash in water. Dehydrate through graded alcohols, clear in xylene & mount in CV mount.