Lamin B1 Antibody (MCE, HY-P80205) Immunofluorescence (IF) Protocol

Product Information

• Antibody: Rabbit monoclonal, unconjugated, targeting Lamin B1 (~66 kDa).
• Applications: Validated for WB, IHC-P, IF-Tissue in human, mouse, rat samples.
• Recommended dilution: 1:100 (optimization advised).
• Antigen retrieval: EDTA-based heat-induced epitope retrieval (HIER, boiling method) recommended.

Immunofluorescence (IF) Staining Protocol

I. Deparaffinization & Rehydration

II. Antigen Retrieval (Critical for nuclear membrane proteins like Lamin B1)

Option 1: Microwave-Based (Tris-EDTA, pH 9.0)

• Heat 650 mL Tris-EDTA buffer (pH 9.0) to boiling (microwave, high power).
• Immerse slides, maintain 95–100°C (low power, 20 min).
• Cool to RT (10 min), rinse with deionized water.

Option 2: Pressure-Based (Citrate, pH 6.0)

• Boil 1 L citrate buffer (pH 6.0) in a pressure cooker (unsealed).
• Seal, heat until steam release, maintain 2 min under pressure.
• Cool slowly, rinse slides to RT.

III. Blocking

• Circle tissue with a hydrophobic barrier pen.
• Apply 10% normal goat serum (NGS) in PBS, incubate 1 hr (RT, humid chamber).

IV. Primary Antibody Incubation

• Dilute Lamin B1 antibody (1:100 in 10% NGS/PBS).
• Apply 100 µL/section, incubate 4°C overnight (or 1 hr RT, if validated).
• Negative control: Omit primary antibody (PBS-only).

V. Secondary Antibody & Nuclear Staining

• Wash 3× 3 min with PBS (pH 7.4).
• Apply fluorophore-conjugated secondary antibody (e.g., goat anti-rabbit IgG) + DAPI (1:2000).
• Incubate 1 hr (RT, dark).

VI. Mounting

• Wash 3× 3 min with PBS.
• Mount with PBS or antifade mounting medium.

Key Considerations

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