protocol_294.txt

Immunofluorescence Microscopy Protocol with Methanol Fixed Cells
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 minutes at 37°C.
At thetime of fixation cells should be ~50% confluent.
Rinse cells briefly in 1X PBS.
Fix cells by incubation with cold 100% methanol for 5‐15 minutes at ‐20°C.
Rinse three times in 1X PBS, 5 minutes each.
Block samples in 5% FBS/PBS for 1 hour at room temperature.
Dilute the primary antibody to the recommended concentration/dilution in 5% FBS/PBS.
For 8 well chamber slides, add 200μl per well and incubate 2‐3 hours at room temperature or overnight at 4°C.
Rinse three times in 1X PBS, 5 minutes each.
NOTE: If using primary antibodies directly conjugated with fluorophores, skip to step 7.
Prepare fluorophore‐conjugated secondary antibody in 5% FBS/PBS according to therecommended manufacturer specification data sheet and add 200μl per well (8 wells) to thechamber slides.
Incubate the samples for 1 hour at room temperature in the dark.
Rinse three times in 1X PBS, 5 minutes each.
Coverslip with anti‐fade mounting medium.
Seal the edges of the coverslip to the slide to prevent movement of the coverslip while imaging.
One sealant that is recommended is a 1:1:1 ratio mixture of vasoline, lanolin and paraffin.
Chamber slides and coverslips Fixation solution: 100% methanol stored at ‐20°C for at least two hours before use.
Antibody dilution solution: 5% FBS in 1X PBS.