Immunohistochemistry or IHC refers to the process of detecting antigens in cells of a tissue section. 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 Pneumococcal 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.
Proteolytic digestion is the hydrolytic breakdown of proteins into simpler, soluble substances such as peptides and amino acids. This method of antigen retrieval uses enzymes to break down the protein cross links created during formalin fixation of the tissue. This process can use several different types of enzymes like pronase, trypsin as well as several others.
This technique has the advantage that enzymes which work at lower temperatures can be used, which would potentially be denatured at the higher temperatures used in heat mediated antigen retrieval. However one disadvantage of this technique is that enzymes, particularly trypsin can end up destroying some epitopes as well as affecting the tissue morphology and so the enzymes require careful monitoring and setting of the optimal incubation time and concentration to reduce these issues.
Pressure cooker heat mediated antigen retrieval involves the application of heat with citrate buffer to unmask the antigen. This involves the use of a pressure cooker for between 1-5 minutes to heat up the slides, which are secured as shown below.
Picture 1 – Slides in pressure cooker
Pressure cooker antigens have the advantage over protease digestion in that the mechanism is heat mediated, so it does not involve the disadvantages involved with protease digestion. Also the pressure cooker is simple to use and takes less time than the use of microwave heat mediated antigen retrieval and the sections are given equal heating treatment, Inconsistency in staining due to hot and cold spots which can occur during microwave treatment is not experienced when using a pressure cooker. However a disadvantage of this technique is the additional health and safety hazards posed to staff by the high temperature required, for example burn injuries. Also the potential for the tissue to be destroyed by the oven is another disadvantage of this procedure.
Microwave antigen retrieval has been shown to be effective in unmasking epitopes in formalin fixed, paraffin wax embedded tissues. This procedure as shown below, similar to the pressure cooker method uses heat to retrieve antigens from normally formalin fixed tissue.
Picture 2 – Slides in microwave oven
The slides are normally deparaffinised in xylene then taken to water through alcohol and spirit. After that the slides are then immersed in buffer. After that they are then microwaved in this solution. Afterwards the slides are then washed in PBS after cooling before going onto normal IHC procedure.One of the problems with microwave antigen retrieval is that it can be difﬁcult to precisely control the heating temperature of the microwave. This however, can be overcome using a scientific microwave which will evenly heat all areas to the same temperature.
To carry out the procedure the general protocol 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.
Positivity -Brown staining
Picture - P53 detected in Immunohistochemistry staining in colorectal cancer