I have been engaged in immunohistochemistry experiments for many years. I have just started doing experiments in this area, and there have been many problems. The most troublesome problem is the non-specific background dyeing problem. After many years of exploration and hands-on experiment, I explored the crux of the problem. For the most common non-specific background dyeing problems in immunohistochemistry, I gave some insights and experience, and I hope to help some researchers. Guidance, I hope that professionals can give criticism and correction. Of course, these experiences and problems are inseparable from the guidance and help of the lilac garden, the small woodworm, the Chinese pathology network and the professional pathologists of the company. Thanks to the help of these websites and professional companies, I have benefited a lot.
Immunohistochemistry is a histochemical technique that uses the basic principles of immunology, the antigen-antibody reaction, to characterize and localize antigens or antibody substances in tissues or cells. The mechanism of non-specific staining of tissues is complex, so it is not easy to control non-specific background staining.
First, the identification of non-specific staining:
It often occurs at the edge of tissue, collagen fibers and plasma exudates, necrotic tissue and poorly fixed tissue centers. It is characterized by diffuse, uniform background staining. It may also be a randomly distributed positive reaction product point, cluster or block.
Second, the reasons for non-specific staining and how to avoid:
Electrostatic adsorption
An antibody is a negatively charged globulin that is easily combined with a positively charged tissue.
The non-immune serum of the second antibody animal is pre-treated with the first antibody to block the charged group on the tissue, thereby removing non-specific binding to the first antibody. This method is the most effective method. If it is not possible, protein blocking may be insufficient or the serum used may be hemolyzed.
2. Endogenous peroxidase
Erythrocytes, granulocytes are particularly high in content and can be identified under the microscope. Do not treat them as positive results.
Paraffin sections were treated with 3% hydrogen peroxide-methanol solution;
3. Washing is not thorough
It is advisable to ensure the time and amount of PBS or TBS rinse in each step. It is best to soak the slices in a dyeing box filled with lotion for a period of time to ensure adequate cleaning. Some people think that it is difficult to ensure that the flushing is clean or not due to the tight budget of the experiment. This is actually a thousand miles of dykes, collapsed in the ant nest.
4. DAB color reaction time is too long
It is best to observe under the microscope a non-specific staining background caused by avoiding too long coloration.
5. Primary antibody dilution
A serum of 1% BSA or a suitable concentration of secondary antibody may be appropriately added to the primary antibody dilution.
The above is the analysis and control of some non-specific staining reasons that I will personally experience, plus online information and help from the company. I hope to provide some help and guidance to you!
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