IHC Troubleshooting: High Background

You can typically expect some amount of background staining during IHC. However, once the level of background staining becomes high enough to obscure important features and structures of the tissue, steps must be taken to reduce it. Background staining can be caused by inappropriate antibody binding or by mistakes during the preparation of the tissue slide. Use this guide to resolve your high background staining:

 

 

Problem

Cause

Solution

Antibody binding incorrectly Excessive primary antibody concentration Titrate antibody to determine optimal concentration
Secondary antibody non-specific binding Run a negative control without primary antibody to assess secondary antibody binding
Use a secondary antibody pre-adsorbed against the immunoglobulin of the sample species
Block your sample with serum from same host as secondary antibody
Antibody cross-reactivity Use Boster primary antibodies guaranteed specific to only their listed targets
Improper preparation of tissue section Insufficient blocking Increase blocking incubation time
Use a different blocking reagent
Insufficient quenching of endogenous peroxidase or phosphatase Quench endogenous peroxidase with 3%H2O2 solution
Quench endogenous alkaline phosphatase with 2mM levamisole
Insufficient biotin or lectin blocking Block endogenous biotin with=streptavidin solution
Block endogenous lectins with alpha-methyl mannoside buffer
Incomplete deparaffinization Increase deparafinization time and use fresh dimethylbenzene
Tissue section too thick for complete reagent penetration Prepare thinner sections
Improper incubation parameters Incubation temperature too high Incubate at 4C
Too much substrate was used Reduce substrate concentration
Reduce substrate incubation time
Choose a substrate with higher signal:noise ratio (e.g. metal-enhnaced DAB)