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 that there were no positive or negative controls included. I'm not sure whether these controls should be set up by me or if it's the responsibility of the pathology department. Should I purchase the necessary reagents myself? Since this is my first time doing this, I'm a bit confused. Could you please explain in more detail? Thank you very much!
Shanghai Jinma Experimental Equipment Co., Ltd. analysis: In immunohistochemical staining, it is essential to ensure that the reaction product observed in the tissue is indeed the result of the specific interaction between the antigen and the antibody. Because many factors can influence the antigen-antibody reaction during immunohistochemistry, proper control groups must be included to interpret the results accurately.
Common control types include:
- Positive Control: This involves using tissue known to express the antigen being tested, following the same procedures as the experimental samples. A positive result confirms that the staining method is reliable and that the reagents are functioning properly. It also helps rule out false-negative results in cases where the test sample is negative. A positive control should always be used in every experiment.
- Negative Control: This uses tissue that does not express the target antigen. The result should be negative, confirming that the staining process is specific and that non-specific reactions are minimized.
- Blank Control: This is the most common type, where the primary antibody is replaced with a buffer like PBS. If the result is negative, it indicates that the staining method is reliable and that background staining from endogenous enzymes or autofluorescence is not interfering.
- Substitution Control: Here, pre-immune serum or non-immune IgG is used instead of the primary antibody. This helps confirm that the signal is due to a specific antibody-antigen interaction rather than non-specific binding. This is especially important when using commercial antibodies.
- Absorption Test Control: In this control, the primary antibody is pre-incubated with the purified antigen. If the result is negative, it shows that the antibody has been effectively blocked and is not reacting with the tissue antigen.
- Self-Control: This involves comparing different structures within the same tissue section. While this can help in some cases, it’s not considered a scientifically valid control on its own. It should always be combined with other controls like blank or substitution controls.
In modern research, it's also common to verify immunohistochemistry results with Western blotting to confirm the presence of the target protein. Proper controls are crucial for ensuring the accuracy and reproducibility of your experiments.
Household Portable Power Station
Household Portable Power Station,Emergency And Energy Storage Power Supply,Generator Energy Storage Ups Power Station,Home Portable Solar Inverter Generator
GuangZhou HanFong New Energy Technology Co. , Ltd. , https://www.gzinverter.com