Ix71 inverted epifluorescence microscope

Transfected HeLa cells were washed briefly in medium and then fixed with either PFA or glyoxal pH 5 for 30 min on ice and another 30 min at room temperature. The cells were labeled with 0.3 μM SNAP‐Cell TMR‐Star (New England BioLabs #S9105S) for 30 min and afterward washed with PBS for 10 min. TMR fluorescence was imaged at the Olympus IX71 inverted epifluorescence microscope.

Sections were imaged with a Leica SP8 laser scanning confocal microscope and LAS X control software or with an Olympus IX71 inverted epifluorescence microscope. For confocal images, each IC tissue section containing retrograde label, 40 × mosaic Z-stacks were taken throughout the entire depth and x–y plane of the IC. The stacks were collapsed into 2D maximum intensity projections and tiled into a single image using LAS X software. ImageJ software was used to adjust the color balance and to draw masks around the edge of the tissue to remove the embedding medium.

HeLa cells adhered to glass coverslips in 24-well plates were transfected overnight using Lipofectamine LTX (Invitrogen, USA) in accordance to manufacturer’s protocol. Cells were washed with PBS, fixed in 4% paraformaldehyde solution (Sigma-Aldrich, USA) and permeabilized in PBS containing 0.1% saponin (Sigma-Aldrich, USA). Cells were labeled for Flag or CD52 in permeabilization solution supplemented with 2% FCS. The endoplasmic reticulum (ER) was stained with rhodamine-labeled concanavalin A (Molecular Probes, USA) diluted in serum-free PBS. Stained and washed coverslips were transferred to the slides and maintained in Dako Cytomation Fluorescent Mounting Medium. The fluorescence images were captured on Olympus IX-71 inverted epifluorescence microscope equipped with Z-axis-motorized objective revolver controlled by Olympus cellSens Dimension software via Olympus Ix2 - UCB Microscope Controller. Twenty slices in Z-stack with 0.3 µm distance in-betweens were captured and deconvolved using cellSens Dimention and Autoquant X3 software, respectively. Recorded data were processed using ImageJ software. Colocalization of GPI-proteins with the ER was estimated through Z-stacks of individual cells and from different areas of the samples as the Pearson coefficient of correlation (1.0 is full colocalization, 0 is no colocalization, and −1.0 is full exclusion).