Immunofluorescence of HeLa cells

1) Fixation

1.1 Methanol fixation

wash cells 3x with PBS to remove the serum

add Methanol (-20°C) for 3 to 5 min

remove the methanol and add PBS quickly

do not let the cells dry out!!!

optional: after the methanol fixation,

20 sec. acetone (-20°C)

cytosol leaks out and a lot of structure is lost

1.2 PFA fixation

remove the medium and rinse cells 3x with PBS

add 3% PFA in PBS (from 16% stock TAAB) for 15 min (careful, do not inhale)

remove PFA and dispose in closed container

immediately add PBS and rinse 3x

replace PBS with 50 mM NH4Cl in PBS for 20 min (quench)

rinse 3x with PBS

cells can be left in the fridge for 3 days max (better 1 day max), if stored for longer, add small amount of PFA and seal with parafilm

these cells can later be permeabilised for 3-5 min with 0.2%TritonX-100 in PBS to analyse internal antigens by immunofluoresscence

1.3 Saponin permeabilisation prior to fixation

filter all saponin solutions, do not store for more than a day

rinse cells 3x with PBS

permeabilse for 3-5 min with 0.02 to 0.2% saponin in Pipes- MTSB (contains K+)

fix immediately for 15 min in 3% PFA/PBS

remove PFA and dispose in closed container

immediately add PBS and rinse 3x

replace PBS with 50 mM NH4Cl in PBS for 20 min (quench)

wash 3x with PBS

before starting with the IF protocol, wash 5 min in saponin/PBS