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IHC Troubleshooting: Nonspecific Staining
Immunohistochemistry (IHC) is one of the many methods that researchers use to visually detect specific antigens in a sample. A variety of issues can arise during the staining step of IHC, such as non-specific staining. Non-specific staining occurs when the primary antibodies bind to proteins other than the target protein, resulting in data unusable for meaningful interpritation.
Here are some tips to reduce non-specific binding when using IHC:
|Improper Sample Preparation||Inadequite Deparaffinization of the tissue section||Increase deparaffinization time|
|Use fresh dimethylbenzine|
|Inadequite quenching of endogenous peroxidases or biotins||Use H2O2 to quench endogenous peroxidase activity|
|Block endogenous biotin with excess free avadin|
|Insufficient blocking||Increase blocking time|
|Section dried out||Avoid allowing your tissue section to dry out|
|Insufficient washing||Increase washing time and number of washes|
|Antibody problems||Antibody contaminated||Affinity purify your antibodies|
|Use Boster high-quality antibodies|
|Excessive primary antibody concentration||Reduce antibody concentration|
Nonspecific staining of human tonsil tissue stained with anti-CD3 Epsilon antibody
Improved staining of CD3 Epsilon in human tonsil tissue