Ab80864

Cells grown on glass coverslips were washed with PBS, fixed for 15 min with 4% formaldehyde, and washed with PBS. After quenching residual formaldehyde with 0.1 M glycine, cells were washed with PBS, permeabilized for 10 min at room temperature with 0.1% Triton X-100, and then washed with PBS. After blocking in 5% fetal bovine serum, cells sequentially were incubated with primary and secondary antibodies diluted in 1% bovine serum albumin (BSA). Apart from the calnexin antibody, which was incubated overnight at 4°C, primary antibodies were incubated for 1 h at room temperature and secondary antibodies were incubated for 45 min at room temperature. Cells were washed in PBS after each antibody incubation. Coverslips were mounted in ProLong Gold antifade reagent (Invitrogen) and analyzed using an LSM 5 PASCAL laser scanning microscope (Carl Zeiss).
The following primary antibodies were used at the indicated dilution: mouse anti-Myc tag (05-724; 1/500; Millipore), rabbit anti-calnexin (2679S; 1/50; New England BioLabs), rabbit anti-Giantin (ab80864; 1/500; Abcam), mouse anti-GM130 (610822; 1/500; BD Pharmingen), sheep anti-TGN46 (AHP500GT; 1/500; AbD Serotec), and rabbit anti-HRV16 2C (this study; 1/500). Secondary antibodies were obtained from Jackson ImmunoResearch (1/200).

Cells were fixed with 4% paraformaldehyde in PBS for 10 minutes, washed three times with PBS, and then permeabilized in 0.2% Triton X-100 in PBS for 5 minutes. After washing three times with PBS and blocking with PBS containing 5% fetal bovine serum (FBS), the cells were immunostained with rabbit anti-flag (2368s, Cell Signaling), rabbit anti-giantin (ab80864, Abcam), mouse anti-pan-Cadherin (ab22744, Abcam), or mouse anti-atp2c1 (WH0027032M1, Sigma) in PBS with 1% FBS at 4°C overnight. The cells were then washed three times with PBS and incubated with mouse anti-calnexin Alexa Fluor 647 (ab202572, Abcam), goat anti-rabbit Alexa Fluor 488 (A11008, Molecular Probes), donkey anti-mouse Alexa Fluor 488 (ab150105, Abcam), donkey anti-rabbit Alexa Fluor 555 (A31572, Molecular probes), and/or goat anti-rabbit Alexa Fluor 350 (A0408, Beyotime) in PBS with 1% FBS at 37°C for 1.5 hours. The slides were mounted under coverslips using ProLong Gold Antifade reagent (Thermo Fisher Scientific), and fluorescence images were obtained using an LSM-710 microscope (Zeiss, Germany) equipped with a 63X/1.4 numerical aperture oil-immersion objective.