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 .
