Immunofluorescence Microscopy Protocol
Grow cultured cells on cover slips or 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 in PBS for 5 min (1/3).
Rinse in PBS for 5 min (2/3).
Rinse in PBS for 5 min (3/3).
Add 0.5% Triton X-100 in PBS and incubate at room temperature for three to five min.
Rinse in PBS for 5 min (1/3).
Rinse in PBS for 5 min (2/3).
Rinse in PBS for 5 min (3/3).
Block samples in 5% FBS in PBS at room temperature for one hour.
Dilute the primary antibody to the recommended concentration/dilution in 5% FBS/PBS.
Add 200 µl per well (8 wells) to the chamber slides and incubate two to three hours at room temperature or overnight at 4°C.
Rinse in PBS for 5 min (1/3).
Rinse in PBS for 5 min (2/3).
Rinse in PBS for 5 min (3/3).
Prepare fluorochrome-conjugated secondary antibody in 5% FBS/PBS according to the recommended manufacturer specification data sheet, and add 200 µl per well (8 wells) to the chamber slides.
Incubate the samples for 1 h, at room temperature, in the dark.
Rinse in PBS for 5 min (1/3).
Rinse in PBS for 5 min (2/3).
Rinse in PBS for 5 min (3/3).
Coverslip with anti-fade mounting medium.
Seal slides with nail polish.