Figure 5A: Immunocytochemical localization of PKCa in isolated intestinal cells. Intestinal cells were isolated by citrate chelation, resuspended in 40 ml of GBSS and cultured with 3 ml of RPMI 1640 in tissue culture dishes containing a cover slip to which were added 0.5 ml of cell suspension. The cells were cultured overnight. The following morning, media were aspirated and replaced with 1 ml of GBSS containing 0.1% BSA. At T = 0 min, either vehicle (0.001% ethanol, final concentration), 130-, 300-, or 650 pM 1,25(OH)2D3 were added. At T = 30 sec, media were aspirated and replaced with 1 ml of 3% paraformaldehyde, 3% sucrose in PBS and the cells are fixed for 20 min. After washing three times with PBS, cells were permeabilized for 5 min with 0.1% Triton X-100 in PBS, washed three times with PBS, and incubated with primary antibody (rabbit anti PKCα, 1/500 dilution) with PBS-0.1% BSA for 30 min, followed by three washes with PBS. Cells were then incubated with fluorescein-conjugated secondary antibody (Jackson Immunoresearch, West Grove, PA) for 30 min, washed three times and mounted on 10% 1 M TRIS in 80% glycerol on microscope slide. Coverslip were blotted, and sealed with nail polish. Arrows indicate the brush border.