IMMUNO-HISTO-CHEMISTRY

Introduction:

Immunohistochemistry (IHC) refers to the process of detecting antigens in cells of a tissue section by using principle of antigen-antibody reactions. Immunohistochemical staining is widely used in the diagnosis of abnormal cells such as those found in cancerous tumors. Specific molecular markers are characteristics of particular cellular events such as proliferation or apoptosis. IHC is widely used in basic research to understand the distribution and localization of biomarkers and differentially expressed proteins in different parts of a biological tissue.

Albert Coons conceptualized and first implement the procedure in 1941

Visualizing and antigen-antibody reaction can be accomplished in a number of ways. Mainly either of the following:

1.       Chromogenic Immunohistochemistry (CIHC): An antibody is conjugated to an enzyme, that can catalyse a colour producing reaction.

2.       Immunofluorescence: The antibody is tagged to a fluorophore such as fluorescein or rhodamine.

Purpose:

1.       IHC can be used to detect mutations in genes that are associated with the development of breast cancer and the risk of developing breast cancer.

2.       IHC can also detect HER2 hormone receptor on the tumor surface. Which rates in on a scale from 0-3⁺.

3.       IHC used to identify replicating cells. Locate cells that are signalling and also helps to locate apoptic cells.

4.       IHC identify the different types of cells in a tissue and detect cellular antigen. Also, it examines the cytoskeletal structure.

Standard IHC Staining Procedure:

1.       3μm Thick sections are mounted on slides coated with adhesives and fixed in incubator for overnight at 37°C or incubated at 60°C for 1hr.

2.       Deparaffinize sections in xylene and grades of alcohol to water.

3.       Quench endogenous peroxidase activity. Incubate slide in hydrogen peroxide block for 10 minutes.

4.       Rinse in buffer wash foe 5 minutes.

5.       Perform antigen retrieval step as desired either using enzymes or heat mediated protocols.

6.       Rinse in buffer wash for % minutes.

7.       Incubate with primary “blocking serum” for 5 minutes.

8.       Rinse buffer wash. This step can be optional.

9.       Incubate with primary antibody for 1hr at room temperature.

10.   Rinse in buffer wash for 10 minutes. (2 changes of 5 min each)

11.   Incubate with secondary link antibody for 20 to 30 minutes

12.   Rinse in buffer wash for 10 minutes.

13.   Apply chromogen substrate for 5 to 10 minutes.

14.   Rinse in distilled water for 5 minutes.

15.   Counter stain with hematoxylin.

16.   Rinse in running tap water.

17.   Dehydrate, clean and coverslip using DPX.

Staining protocol for frozen section:

1.       Snap freeze small pieces of tissue.

2.       Cut frozen sections at 4 microns.

3.       Pic up sections on gelatine chrome coated or PLL coated slide.

4.       Airdry overnight at room temperature (25°C -30°C)

5.       Fix in cold acetone for 20 minutes.

6.       Air dry sections.

7.       Proceed with immune-staining.

For frozen section endogenous peroxidase blocking is not recommended but the use of negative control for the identification of endogenous peroxidase is preferred.

       

Immunohistochemistry Staining Procedure:

Sample Preparation: Proper preparation of the sample is critical to maintain cell morphology,
tissue architecture and the antigenicity of target epitopes, depending on the purpose and the objective, thickness of the tissue is determined. About 4-40μm slicing of tissue is obtain by microtome and mounting on slides. After preparation of sample or slices, the slices deparaffinize in xylene and ascending grades of alcohol to water. Due to fixation and specimen preservation, additional steps are required to make the specific cell epitopes available for antibody binding. Similarly depending on the tissue type and the method of antigen detection endogenous enzymes may need to be blocked.

Selection of paraffin Block: After ample fixation we need to prepare paraffin block of the sample. We use sometime frozen section prepared by cryostat or any other mechanism. Depending on the tissue. We use various temperature for preparation of paraffin block.

Sectioning and Deparaffinization: After the preparation of paraffin block or frozen section, with the help of microtome tissue sectioning is done and then the deparaffinization process of tissue by xylene and ascending grades of alcohol to water. After that the deparaffinized tissue is induced to perform further procedure of IHC that is blocking of endogenous enzymes.

Blocking of Endogenous Enzymes: Before the deparaffinization process we should mount the slide with adhesives poly-l-lysine in an incubator for overnight at 37°C. The next method for blocking of endogenous enzymes we should quench endogenous peroxidase activity and incubate the slide in hydrogen peroxide block for 10 minutes

Antigen Retrieval: Formaldehyde is a commonly used fixative. However, it causes protein linkages, which may result in masking of tissue antigens. Antigen retrieval is pre-treatment prior to immuno-staining used to unmask the antigen. The technique involves use of proteolytic enzyme digestion of heat induced antigen retrieval method.

Primary Antibody: after antigen retrieval incubate the sample with blocking serum for 5 minutes. A respective primary antibody should be added against the respective antigen. This primary antibody is used as a monoclonal antibody which is able to detect only 1 antigen. The antigen is incubated with primary antibody for one hour.

Secondary Antibody: Secondary antibody is mainly used in indirect ELISA test. In this test secondary antibodies provide structural support. Due to the structural bond between 1° and 2° antibody, the washing agent unable to break down the bond between them. A enzyme (HRP) labelled with secondary antibody incubate for 20-30 minutes.

Substrate Chromogen System: A TMB substrate is added to the sample which reacts with Horse reddish Peroxidase (HRP) enzyme and gives an instant colour in an alkaline medium. The intensity of the colour is directly proportional to the concentration of the antigen present in the sample. Apply chromogen substrate for 5-10 minutes.

Counter Staining: After chromogen substrate system a second chemical stain is other applied to provide contrast that helps the primary staining product stand out. The most used counterstain in IHC is Dab, Hematoxylin & Eosin (H&E), Thermo-scientific pierce Hoechst fluorescent stain etc.

Observation: After staining procedure the slide is mounted by DPX and Canada Balsam, therefore the slide observes under the electric microscope at 100x by using cedar wood oil for better visualization.

 

Diagnostic Immunohistochemistry Markers:

Immunohistochemistry (IHC) is an excellent detection technique and has the tremendous advantage of being able to show exactly where a given protein is located within the tissue examined. It is also an effective way to examine the tissue. IHC has become a widely used technique in the neurosciences, enabling researches to examine protein expressions within specific brain structures. Its major disadvantage is that, unlike immunoblotting technique where staining is checked against a molecular weight ladder it is possible to show in IHC that the staining is corresponded with the antigen of interest. For this reason, the primary antibodies must be well validated in a western blot on similar procedure. IHC technique is more widely used in diagnostic pathology for typing tumours, e.g., Immunostaining for e-cadherin (adhesive molecule) to differentiate between DCIS (ductal carcinoma in situ, stains positive) and LCIS (lobular carcinoma in situ, which does not stain positive).

Most recently IHC have been useful in differential diagnosis of multiple forms of salivary gland, head, neck carcinomas.

Following is some test used in Immunohistochemistry –

1.Identification of B cell lymphomas using CD20

2. Identification of T cell lymphomas using CD3

3. CD45 and CD30 used in Hodgkin’s disease

4.CD117 used for gastrointestinal stromal tumours.

5.CD10(CALLA) used for renal cell carcinoma and acute lymphoblastic leukaemia

6.Prostat specific antigen used for PROSTAT gland.

7.Cytokeratins used for identification of carcinomas.

8.Estrogens and Progesterone staining for tumour identification.

 

Chemical Inhibitors for Immunohistochemistry (IHC):

Tumor biology allows for a number of potential intracellular targets. Many tumors are hormone dependent. The presence of hormone receptors can be used to determine if a tumor is potentially responsive to antimonial therapy. One of the first therapies was the antioestrogen, tamoxifen, used to treat breast cancer such hormone receptors can be detected by IHC. Imatinib, an intracellular tyrosine kinase inhibitor was developed to trear chronic myelogenous lukemia, a disease characterised by the formation of a specific abnormal tyrosine kinase. Imatinib has proven effective in tumors that express other tyrosine kinase. Most notably KIT. Most gastrointestinal stromal tumors express KIT. Which can be deleted by Immunohistochemistry.  

Clinical Significance: 

IHC is a method for detecting antigens or haptens in cells of a tissue section by exploiting the principle of antibodies binding specifically to antigens in biological tissues. IHC used for diagnosis of cancer, Specific tumor antigens are expressed denovo or upregulated in certain cancers.

A positive test means that a marker or receptor is bound on the cell during the biopsy or indicates a certain change in the protein of the tumor.