Alexa fluor 488 conjugated goat anti mouse polyclonal antibody

The PK-15 cells were transfected with swine IFITM expression constructs in glass-bottomed coverslips (NEST Biotechnology, Wuxi, China) for 24 h. Then, the cells were fixed with 4% (vol/vol) paraformaldehyde at 4°C for 30 min. Following cell fixation, QuickBlock Blocking Buffer for Immunol Staining (Beyotime, Shanghai, China) was used. All the samples were stained with ANTI-FLAG monoclonal antibody (Sigma-Aldrich) to detect IFITM proteins and anti-LAMP1 monoclonal antibody (Abcam; ab25245) to detect endosomes, and they were then incubated with Alexa Fluor 488 conjugated goat anti-mouse polyclonal antibody (Abcam; ab150113) and Alexa Fluor 594 conjugated goat anti-rabbit polyclonal antibody (Abcam; ab150080). The nuclear DNA was labeled with 4′,6-diamidino-2-phenylindole solution (Beyotime, Shanghai, China). Finally, all the cells were visualized with a Leica DM-IRE2 confocal microscope using a 63× immersion oil objective. Images were captured with the Leica Application Suite advanced fluorescence software (LAS X) and the ImageJ package.

A549 cells were seeded on glass-bottomed coverslips (NEST) and were transiently transfected with IFITM expression constructs. At 24 h after transfection, the cells were fixed with 4% (vol/vol) paraformaldehyde-phosphate-buffered saline (PBS) and were then permeabilized and blocked with QuickBlock Blocking Buffer for Immunol Staining (Beyotime, Shanghai, China). The cell samples were incubated with anti-Flag monoclonal antibody (Sigma-Aldrich, St. Louis, MO, USA) to detect IFITM protein or antilysosome-associated membrane glycoprotein 1 (LAMP1) antibody (Abcam, Cambridge, UK) to detect endosomes, followed by incubation with Alexa Fluor 594-conjugated goat antirabbit polyclonal antibody (Abcam) or Alexa Fluor 488-conjugated goat antimouse polyclonal antibody (Abcam). The nuclear DNA was labeled with Fade-4 6-diamidino-2-phenylindole (DAPI) solution (Beyotime). The cells were finally visualized with a Leica DM-IRE2 confocal microscope.
A549 cells expressing Flag-tagged IFITMs grown in 24-well plates were detected by indirect immunofluorescence using the protocol for confocal microscopy. The cells were finally visualized with a fluorescence microscope.