Immunofluorescence stain of cells on chamber slides

Grow cultured cells on chamber slides overnight, or add appropriate amount of cells to poly-L-lysine coated chamber slides and incubate at least 30 min at 37°C. At the time of fixation cells should be ~50% confluent.

Rinse cells briefly in PBS.

Fix cells by incubation with 4% Paraformaldehyde in PBS, for 15 min (at room temperature).

Rinse the cell cultures in PBS for three minutes. Repeat three times.

Add ice-cold acetone and incubate at -20°C for 10 min.

Rinse the cell cultures three minutes in PBS. Repeat three times.

Block samples in 5% normal serum from same species as secondary antibody in 1% BSA/0.2% Triton X-100/PBS for 1 h at room temperature, or overnight at 4°C.

Dilute the primary antibody to the recommended concentration/dilution in 1% BSA, 0.05% Triton X-100 (PBS).

Add 200 4l per well (8 wells) to the chamber slides and incubate 2 h at room temperature,or overnight at 4°C.

Rinse the cell cultures three minutes in PBS. Repeat three times.

If using primary antibodies directly conjugated with FITC or Alexa Fluor®488, coverslip with anti-fade mounting medium and seal the slides with nail polish.

Prepare fluorochrome-conjugated secondary antibody antibodies in 1% BSA, 0.05% Triton X-100 (PBS) according to the recommended manufacturer specification data sheet and add 200 4l per well (8 wells) to the chamber slides.

Incubate the samples for 1 h at room temperature in dark.

Rinse the cell cultures three minutes in PBS. Repeat three times.

Coverslip with anti-fade mounting medium and seal the slides with nail polish.