Labtekii glass chamber slides

Cells were seeded overnight at 40,000 cells/well onto LabTekII glass chamber slides (Thermo-Scientific, USA). They were washed once with 1×PBS, and either treated or fixed immediately with 4 % paraformaldehyde for 15 minutes. They were washed 3 times with 1×PBS before being incubated for 1 hr at RT with primary antibodies against the respective proteins at a dilution of 1:100 (anti-KIAA0319L and anti-TGN46) or 1:200 (anti-giantin) in IF blocking buffer (PBS with 3% BSA, 1% saponin and 1% Triton X-100). Cells were then washed three times in 1×PBS, and incubated for a further hour in DAPI stain (1:500) and fluorescently-tagged secondary antibodies (Alexa488 anti-mouse and Alexa594 anti-rabbit – Life Technologies) at a dilution of 1:300. Cells were washed a final three times in 1×PBS, and 5 μl of Vectashield (Vector Laboratories Inc, Burlingame, CA) was applied to each slide chamber before a glass cover slip (VWR, USA) was placed over slide to mount samples. Cells were visualized directly with a Zeiss LSM 700 confocal microscope.