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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 (4)
Cell Signaling Technology
Immunohistochemistry Protocol for Paraffin embedded Tissue Sections
Bilibili (China-Accessible Mirrors) ★ 82
Immunohistochemistry on Paraffin-Embedded Sections (CST Demo)
"Step-by-step CST IHC demo on FFPE sections that shows proper antibody incubation steps, helping users spot where over-concentrated primary antibody causes diffuse background."
Cell Signaling Technology ★ 78
Tissue Controls & More for Immunohistochemistry | CST Tech Tips
"Directly addresses positive/negative tissue controls and isotype controls, the exact tools needed to diagnose non-specific background and confirm whether primary antibody concentration is the culprit."
Cell Signaling Technology ★ 72
Better IHC Step 1: Antigen Retrieval
"Focuses on antigen retrieval optimization, a common source of fuzzy/diffuse staining that's often confused with antibody concentration issues in troubleshooting background problems."
Source: xiaohongshu.com ↗
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