Abs20014

At 24 h after transfection, cells were treated for 2 h with 50 μM MG-132 (in DMSO). Cell slides were fixed with 4% paraformaldehyde (in PBS) for 30 min at room temperature (RT), and permeabilized with 0.5% Triton X-100 (in PBS) for 20 min after washing with PBS. Slides were incubated with 5% bovine serum albumin at room temperature for 2 h and then with mouse anti-HA (1:100; Cell Signaling Technology) and rabbit anti-calnexin (1:100; Cell Signaling Technology) for 8 h at 4 °C, respectively. After three washes in PBS, donkey anti-mouse IgG-AlexaFlour 488 antibody (1:200; abs20014, Absin, Shanghai, China) and donkey anti-rabbit IgG-AlexaFlour 594 antibody (1:200; abs20021, Absin) was applied for 2 h. Nuclei were labeled with DAPI (in PBS) for 5 min. Cell slides were imaged using two-photon microscopy (Zeiss NLO 780, Carl Zeiss, Oberkochen, Germany).

Stable OVCA429 cell lines were inoculated on sterilized coverslips in a 12-well cell culture plate. After fixation with 4% paraformaldehyde (5 min), the cells were washed three times with PBS buffer (5 min each time) and permeabilized with 0.5% TritonX-100 for 10 min. 1% BSA was added to block the antigen for 30 min. The primary antibody FSP1 (abs138156, Absin, China; 1:200) and E-cadherin (abs158305, Absin, China; 1:200) were incubated with the samples overnight at 4 °C in a humidified chamber. After incubation, the coverslips were washed with PBS and incubated with Alexa Fluor 594 (abs20021, Absin, China; 1:100) and Alexa Fluor 488 (abs20014, Absin; 1:100) fluorescently labeled secondary antibodies at room temperature in a dark box for 1 h. The nuclei were stained with DAPI before covering the coverslips with anti-fluorescence quenching mounting medium (P0126, Beyotime, China). The slides were observed and recorded using a confocal fluorescence microscope (Olympus, FV3000, Japan).