This image is fig. 6B in PMID:20679433. Exogenous expression of myc-p125A can rescue the dispersed Golgi phenotype in p125A depleted cells. HeLa cells stably expressing GT-GFP were transfected twice with ON-Targetplus SMARTpool siRNA (Dharmacon) against p125A with a 24 hour interval. 48 hour after siRNA transfection, the cells were transfected again to express myc-p125A. At 72 hour after the initial siRNA transfection, the cells were processed for indirect imunofluosecence microscopy to view GT-GFP (green) and myc-p125A (secondary Ab: goat anti-Rabbit Ab labeled with Alexa 555, pseudocolor in red). Technical details: Cells grown on coverslips were washed 2X with PBS supplemented with 1 mM CaCl2 and 1 mM MgCl2 (PBSCM), fixed with 4% paraformaldehyde in PBSCM for 20 min at RT, washed 5X at 5-min intervals using PBSCM, and then permeabilized with 0.1% Saponin in PBSCM for 20 min at RT. The cells were then immunolabeled with appropriate primary antibodies diluted in fluorescence dilution buffer (FDB; PBSCM with 5% FBS and 2% bovine serum albumin [BSA]) for 1 h at RT, washed 5X with 0.1% Saponin PBSCM at 5-min intervals. Secondary antibodies were diluted in FDB and incubated at RT for 1 h, washed again with 0.1% Saponin PBSCM 5X at 5-min intervals, and then 2X with PBSCM. The coverslips were mounted on microscopic slides with Vectashield mounting medium containing DAPI. Confocal microscopy was performed with an Axioplan II microscope (Carl Zeiss, Inc.) equipped with Zeiss confocal scanning optics.
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