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.

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

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 (2)

Cell Signaling Technology ★ 60

Immunohistochemistry Protocol for Paraffin embedded Tissue Sections

"Shows a standard IHC protocol for paraffin sections, useful for ensuring proper technique that avoids localization artifacts."

Cell Signaling Technology ★ 90

Tissue Controls & More for Immunohistochemistry | CST Tech Tips

"Directly addresses antibody specificity validation using tissue and isotype controls, which is the top cause of abnormal nuclear staining."