Hello Sani Aliyu Mohammed
There are several measures that one can take in immunohistochemistry to get quality results.
1. First and foremost, I would use 4μmthickness of tissue section for IHC as thicker tissue sections is likely to produce higher background signals.
2. Antibody binding epitopes can be masked in formaldehyde-based fixation due to cross-linking of amino groups on adjacent molecules. So, antigen retrieval, an additional step to unmask the epitope, may be required. Optimization of the antigen retrieval step would be required to restore antigenicity. Various methods can be used for application of heat such as microwave ovens, heating plate, pressure cookers, autoclaves, and water baths in varying conditions including pH 6–10. I would optimize the antigen retrieval step as an appropriate antigen retrieval would differ from antigen to antigen, and antibody to antibody, and therefore should be determined individually for each antigen and antibody.
3. Protein blocking step is required to reduce unwanted background staining. I would carry out sufficient washing after the blocking step to remove excess protein that may prevent detection of my target antigen. Choosing an appropriate blocking buffer that yields the highest signal to noise ratio would be important for me. For instance, I would not use non-fat dry milk as it contains biotin and would be inappropriate for use with an avidin-biotin complex system. If I would be using peroxidase anti-peroxidase system in the detection step, then blocking of endogenous peroxidase activity would be important. I would make use of diluted hydrogen peroxide as 3% for blocking endogenous peroxidase activity.
4. Selecting a suitable antibody when performing IHC is an important factor. Optimization would be necessary to tune antibody dilution and incubation time. Titration of the antibody and differences between control and testing samples should be carefully considered. I would perform IHC with negative control to disclose any background staining. For an unknown antibody, it would be important to select proper positive and negative control tissues. Interpretation of IHC stain pattern in control tissues should be done carefully.
5. There are various methods that can be used as detection system such as avidin-biotin complex method, labeled streptavidin biotin method, phosphatase anti-phosphatase method, polymer-based detection system, and tyramine amplification system. Depending on the type of tissue, the detection system could be selected. For instance, AP-based detection system is preferred for tissues rich in endogenous peroxidase, such as bone marrow, or peroxidase based-detection system may be used for tissues containing endogenous APs, or biotin-free synthetic polymer system could be used for tissues with high endogenous biotin such as liver and kidney.
6. Hematoxylin is the most used counterstain which would provide contrast to the chromogens for better discrimination of the target signal. It would help to identify the cell type and exact localization of the immuno positives.
Best.
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