Immunofluorescence and Bodipy staining of lipid droplets

From LipidomicsWiki

Immunofluorescence and Bodipy staining of lipid droplets (A431, HeLa cells)

Reagents:

- PBS (phosphate buffered saline)
- PBS-4% Paraformaldehyde
- Blocking buffer (BB): PBS-0.1%Saponin-0.2%BSA
  for 50 ml use 0.05g Saponin
                      0.10g Albumin – Fraktion V
                      Add to 50 ml with PBS
                      Use within 24 h
- Primary antibodies and fluorescent 2ndary antibody conjugate
- Mowiol mounting medium (Calbiochem) containing 50 mg/ml 1,4-diazabicyclo-[2,2,2]octane (DABCO, Sigma)

Procedure:

• wash cells 1x with PBS
• incubate cells 30’ with PBS-4% Paraformaldehyde
• wash 2x with PBS
• incubate 30’ with BB
• incubate 60’ with first antibody diluted in BB
• wash 3x 10’ with BB
• incubate 60’ with 2ndary antibody conjugate diluted in BB
- include in the 2ndary antibody dilution 1:1000 of Bodipy 493/503 (Molecular Probes; 50 mM stock in DMSO)
• wash 3x 10’ with BB
• rinse 2x briefly with PBS
• incubate 10’ in PBS at room temperature
• wash 1x with distilled water
• mount on object glass using 5 µl Mowiol-DABCO

see discussion