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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 ★ 78
Immunohistochemistry Protocol for Paraffin embedded Tissue Sections
"Comprehensive CST protocol video with associated guide on successful IHC, providing reference standards for proper antibody handling and storage to prevent degradation."
Cell Signaling Technology ★ 92
Tissue Controls & More for Immunohistochemistry | CST Tech Tips
"Directly addresses antibody validation and positive/negative tissue controls, which is critical for diagnosing the failure of non-staining positive controls."
Bilibili (China-Accessible Mirrors) ★ 75
Immunohistochemistry on Paraffin-Embedded Sections (CST Demo)
"Step-by-step CST expert demonstration covers the complete protocol including proper reagent preparation and handling, essential for identifying where antibody storage/degradation issues originate."
Source: xiaohongshu.com ↗
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