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Immunohistochemistry (IHC) severe

Abnormal nuclear staining (wrong subcellular localization)

Symptom
Cell nuclei show strong staining when they shouldn't, or expected positive nuclei show no staining; nuclear/cytoplasmic localization is wrong.
Common Causes
  1. 1 Poor antibody specificity
  2. 2 Antigen retrieval was too strong, exposing nuclear epitopes inappropriately
  3. 3 Antibody recognition site is unclear
  4. 4 Antibody cross-reactivity
Solutions
  1. 1 Switch to a more specific antibody (validated by KO/KD control)
  2. 2 Adjust antigen retrieval conditions; reduce over-retrieval
  3. 3 Check antibody datasheet to confirm intended localization
  4. 4 Add washing steps to reduce non-specific binding
Related Video (3)
Cell Signaling Technology
Immunohistochemistry Protocol for Paraffin embedded Tissue Sections
Cell Signaling Technology ★ 88
Tissue Controls & More for Immunohistochemistry | CST Tech Tips
"Tech tip on positive/negative tissue and isotype controls directly addresses verifying antibody specificity and diagnosing wrong subcellular staining."
Bilibili (China-Accessible Mirrors) ★ 72
Immunohistochemistry on Paraffin-Embedded Sections (CST Demo)
"CST paraffin IHC demo covering antigen retrieval and staining steps gives proper context for where localization artifacts arise."
Source: xiaohongshu.com ↗
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