Ab50595

HEK293 cells expressing corin were treated with CHX for 4 hr, fixed with 3% paraformaldehyde, permeabilized with 0.2% Triton X-100, and imminostained with antibodies against V5 (1:1000), PDI (1:200, Abcam, ab3672) or TGN46 (1:200, Abcam, ab50595) and Alexa Fluor-594 or 488-labeled secondary antibody (1:1000, Thermo Fisher, A-21203; A-11008). In controls, the primary antibody was replaced by mouse (Thermo Fisher, MA110419) or rabbit (Sigma, I5006) IgG. The stained cells were examined under a confocal microscope (Leica DM2500) and images were analyzed by ImageJ software.

CRISPR-mutations third-instar larvae raised at 25°C were harvested, dissected in PBS 1×, fixed in 4% paraformaldehyde for 15 min and washed twice in PBS containing 0.3% Triton X-100 (Sigma-Aldrich). Dissected larvae were probed with anti-GM130 (Abcam, ab30637, rabbit, 1:1000) overnight at 4°C, then washed 3 times with PBS plus 0.3% Triton X-100 and incubated with Alexa Fluor 555 anti-rabbit antibody (Molecular Probes Invitrogen, 1:500). Larvae were washed 3 times with PBS and mounted on coverslips using Mowiol (Sigma-Aldrich).
HeLa cells transfected as indicated above were fixed in PBS containing 4% paraformaldehyde for 15 min, incubated with 50 mM NH₄Cl for 20 min, permeabilized with 0.1% Triton X-100 in PBS for 3 min and then blocked with 2% BSA and 0.2% gelatin for 30 min. Antibodies used were anti-GM130 (BD, #610822, mouse, 1:1000); anti-TGN46 (Abcam, ab50595, rabbit, 1:200). Alexa Fluor 555 conjugated goat anti-mouse and Alexa Fluor 647 conjugated goat anti-rabbit (Molecular Probes Invitrogen) were applied for 1 h at room temperature. Coverslips were mounted using Mowiol (Sigma-Aldrich).
All confocal images were acquired using a Leica TCS SP5 II confocal microscope, equipped with a PlanApo 100×/1.4 Oil objective, using a 543 nm laser line. Confocal microscopy imaging was performed at 1024 × 1024 pixels per image, with a 200 Hz acquisition rate.