Knowledge Base › Troubleshooting Atlas › IHC
IHC background staining (non-specific)
Also known as: high background, dirty IHC
What it looks like
Diffuse non-specific brown (or chromogen) staining across the section obscuring specific signal.
Where it usually appears: Inadequate blocking, over-strong antibody, or insufficient washing.
Likely causes
- Insufficient protein/peroxidase block
- Primary antibody too concentrated or incubated too long
- Inadequate washing between steps
- Endogenous peroxidase/biotin not quenched
- Sections dried during the run
Immediate checks
- Optimise (increase) blocking step
- Titre the primary antibody to a higher dilution
- Increase wash steps/duration
- Include peroxidase (and biotin if relevant) block
Corrective action
- Repeat with optimised dilution, blocking and washing
- Run a known positive and negative control
Preventive action
- Validate each antibody dilution/protocol
- Standardise antigen retrieval
- Run controls with every batch
This atlas is for educational purposes for laboratory professionals. It does not replace your institutional SOPs, manufacturer instructions, validated protocols or pathologist judgment. Adapt any action to your laboratory and equipment.
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