Fixation and Immunostaining of Cells

This is modification from a previously published protocol described in [29 (link)]. Cells in 25 μl growth medium were rapidly fixed by the addition of 25 μl aldehyde in PBS to achieve a final concentration of 4% FA and 0.2% GA (glutaraldehyde); fixation was allowed to proceed for 15 min at room temperature (20–24 °C) before rinsing three times with PBS containing 50 mM NH₄Cl. Slides were then placed on a metal plate in a deep ice bath and chilled for at least 2 min. All subsequent steps were performed on ice, with all solutions pre-chilled. Cells were blocked and permeabilized for 45 min with a solution of buffer A containing 5% (v/v) NGS (normal goat serum), 50 mM NH₄Cl and 0.5% saponin. 100 nM GST–PH-PLCδ1 was included at this stage when the protein was used as a probe for PtdIns(4,5)P₂, and GST-tagged protein was then removed by two rinses with buffer A. Primary antibodies were applied in buffer A with 5% NGS and 0.1% saponin for 1 h. After two washes in buffer A, a 45 min incubation with secondary antibody in buffer A with 5% NGS and 0.1% saponin was performed. Slides were then rinsed four times with buffer A, and cells were post-fixed in 2% FA in PBS for 10 min on ice, before warming to room temperature for an additional 5 min. FA was removed by three rinses in PBS containing 50 mM NH₄Cl, followed by one rinse in distilled water. Wells were then dried, covered with 3 μl ProLong Gold (Invitrogen) supplemented with 1 μg/ml DAPI (4′,6-diamidino-2-phenylindole) and covered with 22 mm×22 mm glass cover slips (No. 1 thickness, Scientific Laboratory Supplies), and sealed with nail varnish.