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: 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
Overstaining of mouse liver tissue stained with anti-SC10A1 antibody

Overstaining of mouse liver tissue stained with anti-SC10A1 antibody

improved staining of mouse liver tissue using Boster troubleshooting tips

Improved staining of VSC10A1 in mouse liver tissue