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)

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Immunohistochemistry on Paraffin-Embedded Sections (CST Demo)

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

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Better IHC Step 1: Antigen Retrieval

"Focused video series on antigen retrieval optimization, the primary determinant of nuclear membrane permeabilization success"

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Complete IHC Immunohistochemistry Protocol for Beginners

"Comprehensive protocol walkthrough that includes proper permeabilization procedures needed to access nuclear compartments"