Large-scale immunofluorescence

This protocol describes our method for preparing cells for immunofluorescence, in which all incubations and washes are performed in microtiter dishes. Two dishes can be processed simultaneously. The last step is to transfer each sample to a well on a polylysine coated 8-well slide for visualization. The protocol can also easily be adapted for preparing cells for immunofluorescence in microfuge tubes.The antibody cleanupprotocol is now a seperate documentProtocol Annotations refer to notes in list given below For analysis of fusion proteins expressed during vegetative growth, inoculate yeast strains into 70 ul of YPD in round-bottomed microtiter wells and grow for ~12 hrs1 at 30o C on a vortex shaker (set on the lowest setting) or orbital platform shaker (240 r.p.m.). Centrifuge microtiter dishes for 5 minutes at 500 X g to pellet cells. Fix cells by adding 1/10 volume of 37% formaldehyde and incubating at room temperature for 15 minutes to 1 hour 2 without shaking. After fixation, pellet cells. Wash cells 3 times with 100 ul Solution A , pelleting between each wash, and finally resuspend in 100 ul of Solution A containing 0.1% beta-mercaptoethanol, 0.02% glusulase and 5 ug/ml zymolyase 100T. After incubation at 37o C for 60 minutes, wash cells3 twice with 100 ul PBS and once with PBS containing 0.1% NP404 . (Many cells are lost at this step.) Add primary antibodies5, 6 to each well in 30 ul of PBS plus 0.1% BSA (bovine serum albumin). Incubate overnight7 at 4o C with very gentle shaking. Wash cells once with PBS, once with PBS plus 0.1% NP40, and then again with PBS. Resuspend cells in 30 ul of PBS plus 0.1% BSA containing the secondary antibody8 , and incubate for 4 hours at room temperature or overnight at at 4o C with very gentle shaking. Wash cells as in step 5, with the addition of an extra wash in PBS plus 0.1% NP40. Finally, resuspend cells in 30 ul PBS. Transfer 15 ul to individual wells on a polylysine-treated 8-well slide9 . Allow cells to settle for 15 minutes or longer (use a humid box to prevent drying out). Gently aspirate off excess solution and immediately add 4 ul of mounting solution to each well. Add a coverslip, seal edges with nail polish and store slides at -20o C in the dark. Solutions Solution A1.2 M sorbitol, 50 mM KPO4, pH 7.0 PBS 150 mM NaCl, 50 mM NaPO4, pH 7.4 Mounting solution70% glycerol/30% PBS containing 2% n-propyl gallate and 0.25 ug/ml Hoechst Primary antibodies rabbit anti-beta-gal antibody (Cappell Laboratories #0631-0002, at 1:200 dilution) rat monoclonal anti-yeast tubulin (YOL1/34; Sera-lab, at 1:100 dilution of monoclonal supernatant) mouse monoclonal anti-HA (MMS101R, BAbCo, at 1:200 dilution) mouse monoclonal anti-HA (12CA5, Boehringer, at 1:200 dilution) rabbit polyclonal anti-HA (RS1010C, BAbCo, at 1:200 dilution)Secondary antibodiesTexas Red-conjugated anti-rabbit (Jackson Labs, 1:200 dilution) FITC-conjugated donkey anti-rat (Jackson ImmunoResearch Laboratories, 1:100 dilution) Cy3-conjugated goat anti-mouse (Jackson Labs, 1:300 dilution)Important Notes Dilution of overnight cultures and further incubation may be necessary to ensure the culture is in log phase, not stationary. Shorter fixation times (15-30 minutes) are recomended if anti-HA antibodies are to be used, as t