Immunohistochemical staining results need to set up a control group to make a correct judgment - Database & Sql Blog Articles

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Abstract: I did TNF-α immunohistochemistry on rat tissue, which was sent to the hospital pathology department for processing. Now that HE and immunohistochemistry slides are ready, I realized there were no positive or negative controls. I'm confused—should these controls be set up by the pathology department, or is it my responsibility? Do I need to purchase the reagents myself? I haven't done this before, so I'm really unsure. Could you please explain in detail? Thanks a lot!

Shanghai Jinma Experimental Equipment Co., Ltd. analysis: In immunohistochemical staining, it's essential to ensure that the reaction product observed in the tissue is indeed the result of the specific interaction between the antigen and the corresponding antibody. Since many factors can influence antigen-antibody reactions during immunohistochemistry, proper control groups are necessary to interpret the results accurately.

Commonly used controls include:

1. Positive Control: This involves using tissue known to express the target antigen and performing the same procedure as the test sample. A positive result confirms that the staining method is working correctly and that the reagents are functional. It also helps rule out false negatives in cases where the test sample appears negative. A positive control should be included in every experiment.
2. Negative Control: This uses tissue that does not express the target antigen. The result should be negative, confirming that any staining seen in the test sample is not due to non-specific binding or cross-reaction.
3. Blank Control: This is the most common type of control, where the primary antibody is replaced with a buffer like PBS. The result should be negative, indicating that the staining method is reliable and ruling out background staining from endogenous enzymes or autofluorescence.
4. Substitution Control: Here, pre-immune serum from the same animal as the primary antibody is used instead of the primary antibody. This helps determine whether the observed staining is due to a specific antibody-antigen interaction rather than non-specific effects of the serum. However, commercial antibodies may not always require this type of control.
5. Absorption Test Control: This involves pre-incubating the primary antibody with excess purified antigen. If the antibody is fully blocked, the immunohistochemical staining should be negative, confirming the specificity of the antibody.
6. Self-Control: This refers to comparing different structures within the same tissue section. While this can provide some insight, it's not considered scientifically rigorous on its own. It’s best to use additional controls like blank or substitution controls for more accurate interpretation.

In modern research, immunohistochemical results are often validated using Western blotting to confirm the presence of the target protein. Including these controls ensures the reliability and reproducibility of your findings. If you're new to this process, don’t worry—setting up proper controls is a crucial step that will help you avoid errors and improve the quality of your data.