Remove culture media from dishes and replace with 2ml PBS.
Replace PBS with 2ml of 2% Fixative and fix for 10min at RT.
Transfer all coverslips to a submerged (in PBS) Coors Holder. Place jar on rotating platform and agitate gently for 10min. Repeat wash twice more in fresh PBS for 10min each.
Transfer holder to ice-cold methanol for 3 min at –20°C, if permeabilization is required. (Or use a mild detergent (0.1 – 0.3% Triton) Permeabilization is not recommended for plasma membrane proteins.)
Transfer holder to PBS and wash samples in PBS for 3x10minwith agitation. (Prepare antibodies in 10% serum from the host of the secondary antibody)
Transfer coverslips, one at a time, to incubation box and add 250µl of appropriate antibody or PBS/10% serum (controls).
Place the lid on the box and incubate in primary antibodies overnight at 4°C.
Remove one coverglass from box, and blot excess antibody using a paper towel. Dip coverslip in a beaker of PBS for 5s, then place it into a Coors holder in PBS.
Wash samples in PBS for 3x10min with agitation. (Prepare secondary antibodies in 10% serum)
Transfer coverslips, one at a time, to incubation box and add 250µl of appropriate secondary antibody or PBS/10% serum.
Incubate in appropriate secondary antibodies for 1hr. at RT.
Remove one coverglass from box, and blot excess antibody using a paper towel.Dip coverslip in a beaker of PBS for 5s, then place it into a Coors holder in PBS.
Wash samples in PBS for 3x10min with agitation.
Transfer coverslips, one at a time, to incubation box and add 250µl of 300nM DAPI. Start a timer on the first coverslip. Incubate each coverslip for 2min.
One coverglass at a time, allow DAPI to run off onto a paper towel, then place coverglass back into Coors Holder in PBS. Rinse for 2min with agitation.
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