IHC Technical Resources

Protocols, optimization tips,
troubleshooting guides,
and more for IHC.

Troubleshooting guides

Troubleshooting guides

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handbooks for IHC, Western
blot and ELISA for FREE.

Tip: For comprehensive troubleshooting resources, download our troubleshooting guides.

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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:

Problem Cause Solution
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

Nonspecific staining of human tonsil tissue stained with anti-CD3 Epsilon antibody

improved staining of human tonsil tissue using Boster troubleshooting tips

Improved staining of CD3 Epsilon in human tonsil tissue