Hoatlin:IFA Protocol

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Immunofluorescence Protocol (HeLa cells)

Grow adherent cells (e.g. seed at 20,000 cells/mL in a 4 well) on chamber slides or cover slips until the time of harvest.

• Wash cells 3 x 5 min with phosphate-buffered saline (PBS)

• Fix cells onto slides/cover slips with 3.7% formaldehyde in PBS for 5 min @ 4˚C)

• Permeabilize cells with PBS containing 0.1% Triton X-100 for 3 min @ 4˚C

• Block with 7.5% BSA in PBS (1 h @ RT or overnight @ 4˚C)

• Incubate with primary antibody diluted in PBS containing 0.5% BSA (1-2 h @ RT or overnight @ 4˚C)

• Wash cells 3 x 5 min (RT)

• Incubate with secondary antibody for 1 h @ RT (we use AlexaFluor antibodies, Molecular Probes)

• Counterstain the nuclei with PBS containing 10 ng/ml Hoechst 33342 (Molecular Probes) for 3 min @ RT

• Mount the cells with anti-fade mounting solution (Vectashield, Vector Laboratories)

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