Immunofluorescence Protocol
important recommendation: avoid drying out coverslip after cell incubation since surface
tension will tear cells even after fixation
coat coverslips (cs), placed on lid of a 24-well dish, with fibronectin (0.5 - 1 µg/cs)
diluted in PBS or Col IV/CB3 fragment (0.1 - 0.5 µg/cs) in water by drying down with
fan.
rehydrate with PBS
resuspend cells after brief trypsin treatment in DMEM for staining or
DMEM/1 - 10 % CS (when incubating overnight):
1:50 split of a confluent 10 cm dish
to prevent protein synthesis if necessary, incubate cells for 1 h in DMEM containing
25 µg/ml cycloheximide before trypsin treatment, maintain cyclohex. throughout the
experiment
incubate cells with cs in 24-well dish for 2 - 3 hours at 37° C
at this point prepare 1 l of complete PBS (PBS containing 1 mM of Ca 2+ and Mg2+
each)
prepare 4% paraformaldehyde (50 ml H20 + 10 ml 10x PBS + 40 ml 10%
paraformaldehyde + 0.5 ml Ca2+/Mg2+ 100 mM), keep on ice
prepare 0.2% NP-40 in cPBS
after incubation, remove cs from 24-well dish using a syring needle and tweezers, place
in porcelain rack, coated sides facing you.
wash cs 3 times in cPBS (conveniently in glass trays)
fix cells in 4% paraformal. for 5 min., wash 3x in cPBS
lyse cells by incubating cs in 0.2% NP-40 for 5 min.
prepare first antibody sltn: 1:200 for polyclonal or ascites mAb in cPBS containing 10%
NGS (goat serum) or 90% mAb supernatant/10% NGS
wash 3x, tip edge of cs in NP40 sltn to reduce surface tension
place cs on lid of 24-well dish, add 25 µl of first antibody sltn per cs, do one cs at once
incubate at 37° C for 30 min on a wetted paper towel in covered plastic tray
prepare secondary antibody sltn: rhodamin rabbit anti mouse IgG for mouse mAb,
1:500 in PBS/10% NGS or fluorescein goat anti rabbit 1:500 in PBS/10% NGS for
rabbit polyclonal serum
put cs in porcelain rack, wash 3x with cPBS,
tip edge in NP40, place on dish lid, add 25 µl of 2nd antibody sltn, incubate at 37° C as
above
wash 3 x with cPBS, wash once with dd water
place one drop of aquamount on glass slides and mount cs upside down (cells facing
aquamount) , keep for 2 h at 4° C
place one drop of immersion oil on cs, bring lense of i.f. microscope cautiously in
contact with oil and check coverslip with approriate wavelength