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Immunohistochemistry (IHC) critical

False negative — no staining at all

Symptom
Even the positive control shows no staining; expected positive regions are completely blank; the experiment failed.
Common Causes
  1. 1 Antibody is degraded or stored incorrectly
  2. 2 Antigen retrieval failed
  3. 3 Detection reagent has expired
  4. 4 Protocol steps were missed or done in wrong order
Solutions
  1. 1 Use a fresh antibody with proper storage (aliquot, -20 °C)
  2. 2 Re-optimize antigen retrieval conditions (try a different buffer or longer time)
  3. 3 Check expiry dates of all reagents (DAB, secondary antibody, blocking serum)
  4. 4 Strictly follow the protocol step by step; include a positive control slide every time
Related Video (3)
Cell Signaling Technology ★ 72
Immunohistochemistry Protocol for Paraffin embedded Tissue Sections
"Comprehensive CST IHC protocol video with linked troubleshooting guide; essential reference for proper antibody storage and standard protocol to identify where failure originated"
Cell Signaling Technology ★ 92
Tissue Controls & More for Immunohistochemistry | CST Tech Tips
"Directly addresses antibody validation and positive/negative tissue controls—the core diagnostic approach for detecting antibody degradation or failure in IHC"
Bilibili (China-Accessible Mirrors) ★ 78
Immunohistochemistry on Paraffin-Embedded Sections (CST Demo)
"Step-by-step CST protocol demonstration with explicit focus on proper reagent handling and antigen retrieval, providing baseline for identifying protocol deviations that cause no staining"
Source: xiaohongshu.com ↗
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