IHC Troubleshooting: Overstaining

Overstaining occurs due to excessive development of signal in the sample. This causes the sample to become saturated, reducing contrast. Overstained samples can appear blurry, diffuse, or monochromatic. Overstaining can prevent accurate visualization of tissue structures and inhibit useful detection of protein localization.

Here are some tips to reduce overstaining when using IHC:

 

Causes

Solutions

Primary antibody too concentrated Dilute primary antibody solution
Perform a titration to determine optimal antibody dilution
Excessive primary antibody binding Reduce the length of the incubation step
Incubate in a cold room at 4C
Detection substrate incubation time too long Allow less time for signal development after adding the detection substrate
Insufficient washing Increase number and time of washes