Nuclear protein not detected due to insufficient permeabilization

Symptom

No staining observed when targeting nuclear proteins. Antibody cannot access nuclear compartment despite proper antibody concentration and incubation time.

1. 1 Antibody cannot penetrate nuclear membrane due to insufficient permeabilization
2. 2 Weak or absent permeabilizing agents in blocking and antibody buffers
3. 3 Nuclear envelope remains intact preventing antibody access to nuclear epitopes

1. 1 Add strong permeabilizing agent like Triton X to blocking buffer
2. 2 Include Triton X in antibody dilution buffer
3. 3 Increase permeabilization time for better nuclear penetration
4. 4 Use concentration of 0.1-0.5% Triton X-100 for nuclear targets

Related Video (3)

Bilibili (China-Accessible Mirrors) ★ 85

Immunohistochemistry on Paraffin-Embedded Sections (CST Demo)

"Explicitly covers antigen retrieval and permeabilization steps critical for nuclear protein access in paraffin-embedded sections"

Cell Signaling Technology ★ 82

Better IHC Step 1: Antigen Retrieval

"Focused series on antigen retrieval optimization, the direct solution to insufficient permeabilization blocking nuclear antibody penetration"

Bilibili (China-Accessible Mirrors) ★ 72

Complete IHC Immunohistochemistry Protocol for Beginners

"Comprehensive protocol walkthrough from sample preparation through staining includes permeabilization steps necessary for nuclear protein detection"