IHC Troubleshooting:

NON-SPECIFIC STAINING

This guide serves as a checklist for the possible causes and solutions with respect to non-specific staining from the IHC assays.

How To Resolve Non-Specific 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 interpretation.

What Causes Non-specific Staining in IHC?

There are two main causes of non-specific staining in IHC namely ;

  • Improper Sample Preparation
  • Antibody problems

Examples of Non-specific Staining


Non-specific staining image

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


improved staining image

Improved staining of CD3 Epsilon in human tonsil tissue

Issues Related To Sample Preparation


Causes Solution
Inadequate Deparaffinization of the tissue section Increase deparaffinization time
Use fresh dimethyl benzene
Inadequate quenching of endogenous peroxidases or biotins Use H2O2 to quench endogenous peroxidase activity
Block endogenous biotin with excess free avidin
Insufficient blocking Increase blocking time
Section dried out Avoid allowing your tissue section to dry out
Insufficient washing Increase the washing time and number of washes

Issues Related To Antibodies


Causes Solution
Antibody contaminated Affinity purify your antibodies
Use Boster high-quality antibodies
Excessive primary antibody concentration Reduce antibody concentration