Diamidino 2 phenylindole dapi

NSCLC cells (4 × 10⁴ cells/well) were seeded in 6-well plates and incubated in normoxic and hypoxic incubators, respectively for 48 h. After washing three times, the cells were fixed using paraformaldehyde for 1 h, and then blocked using 5% FBS for 1 h. Subsequently, anti-LC3 (1:200, CST) and anti-p62 (1:200, CST primary antibodies were added and incubated overnight at 4 °C. The next day, after incubation with the Alexa-conjugated anti-mouse secondary antibodies (Alexa488, Invitrogen) and Alexa-conjugated anti-rat secondary antibody (Alexa555, Invitrogen) at room temperature for 1 h, the cells were stained using diamidino-2-phenylindole (DAPI, Solarbio, Beijing, China) for 5 min. Subsequently, a confocal microscope was used to observe the cells and obtain data.

In this assay, transwell permeable supports were employed, with a polycarbonate membrane which was coated with Matrigel (Corning Inc., USA) (for invasion tests) or without Matrigel (for migration tests). 24 hours after transfection, the cells were seeded in the upper layer in 100 μL serum-starved RPMI-1640, and then, 600 μL of medium with 10% FBS was added to the bottom layer. After 48 hours of incubation, the cells on the top layer of the transwell chamber were wiped with cotton swabs, and methanol was used to fix the cells in the lower surface for 10 minutes, and subsequently, cells were stained with 10 μg/mL of diamidino-2-phenylindole (DAPI) (Solarbio, Beijing, China) at room temperature for 15 minutes. A 200x fluorescence inverted microscope (Mshot, China) was used to photograph and count the stained cells in ten randomly selected fields.

CRFK cells infected with FPV isolate (MOI = 0.1) in 96-well plates were treated with cold 80% (v/v) acetone for 15 min, washed with PBS three times, exposed to a mouse anti-CPV antibody (Abcam, Cambridge, United Kingdom) at 37°C for 1 h, and then treated with an Alexa Fluor 488-conjugated goat anti-mouse IgG H&L antibody (Abcam, Cambridge, United Kingdom). Then, diamidino-2-phenylindole (DAPI) (Solarbio, Beijing, China) dye was added for fluorescent staining of cell nuclei. FPV infected cells were observed under a fluorescence microscope (ZEISS, Oberkochen, Germany) after being washed with PBS; cells evidencing intranuclear fluorescence were considered infected with FPV.