Knowledge Base › Troubleshooting Atlas › IHC
IHC false negative (no staining where expected)
Also known as: loss of signal, negative control tissue negative
What it looks like
Expected positive tissue shows little or no specific staining; the positive control may also be negative.
Where it usually appears: Pre-analytical problems (fixation, decalcification), retrieval failure, or reagent errors.
Likely causes
- Under- or over-fixation, or prolonged cold ischaemia
- Acid decalcification degrading antigen
- Antigen retrieval inadequate or wrong method/pH
- Antibody too dilute, expired, or a skipped step
- Wrong retrieval for the target
Immediate checks
- Check the positive control ran correctly
- Review fixation time and decalcification method
- Confirm the correct antigen-retrieval method/pH
- Verify antibody, dilution and that no step was missed
Corrective action
- Repeat with the validated retrieval and a fresh antibody aliquot
- If pre-analytical, note the limitation in the report
Preventive action
- Standardise fixation (time and NBF)
- Use EDTA/gentle decalcification where antigen-sensitive
- Validate retrieval per antibody; run controls 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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