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Immunohistochemistry (IHC) severe

Strong positive background / non-specific staining

Symptom
Whole slide appears brown/diffuse stained; negative control also shows signal; impossible to distinguish true positive signal.
Common Causes
  1. 1 Primary antibody concentration too high
  2. 2 Antigen retrieval was too aggressive or wrong protocol
  3. 3 Inadequate blocking
  4. 4 High residual HRP activity in detection system
  5. 5 DAB color development time too long
Solutions
  1. 1 Reduce primary antibody concentration or shorten incubation time
  2. 2 Optimize antigen retrieval (time, temperature, buffer)
  3. 3 Strengthen blocking (use normal serum + BSA appropriate for secondary antibody host)
  4. 4 Use high-quality secondary antibody and detection system
  5. 5 Shorten DAB development time; monitor under microscope
Related Video (3)
Cell Signaling Technology ★ 70
Immunohistochemistry Protocol for Paraffin embedded Tissue Sections
"A full protocol video that shows standard dilution steps and overall workflow to avoid over-concentrated primary antibody."
Cell Signaling Technology ★ 90
Tissue Controls & More for Immunohistochemistry | CST Tech Tips
"Directly explains the use of controls to distinguish true signal from background, addressing the negative control issue in this failure."
Cell Signaling Technology ★ 75
Better IHC Step 1: Antigen Retrieval
"Specifically troubleshoots fuzzy or diffuse IHC staining, which aligns with the background staining problem."
Source: xiaohongshu.com ↗
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