Effect of caffeine and reduced temperature (20 degrees C) on the organization of the pre-Golgi and the Golgi stack membranes

At the end of the incubations, the cells were fixed for 30 rain at room temperature with 3% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4, followed by three washes with PBS. The cells were then permeabilized for 30 min with 0.05% Triton X-100 in PBS at room temperature followed by three washes with PBS containing 0.1% BSA. The primary antibodies were always incubated for 30 rain at 37~ whereafter the cells were washed with PBS containing 0.1% BSA. Incubation conditions for the second antibodies were the same as for the primary antibodies. Finally, the cells were washed and mounted in 75 % glycerol, pH 8.5, on microscope objective glasses and viewed with Zeiss Lab 16 fluorescence microscope with 100x oil immersion objective. Film used for photography was TMAX 400 ASA (Eastman Kodak Co., Rochester, NY).