A1r a1 laser scanning confocal microscope

To assess the colocalization of VP2, p62, and LC3, DF-1 cells were seeded in confocal dishes and cotransfected with Flag-VP2 and Myc-p62 with or without EGFP-LC3. After 24 to 36 h of transfection, cells were fixed using 4% paraformaldehyde for 10 min and permeabilized with 0.2% Triton X-100 for 5 min at room temperature. The fixed cells were incubated with primary anti-Flag and anti-Myc antibodies diluted in 5% skimmed milk for 4 h at 37°C or at 4°C overnight. The cells then were washed with PBS four times and incubated with the corresponding secondary antibodies for 2 h at 37°C. The cells next were stained with 4ʹ,6-diamidino-2-phenylindole (DAPI) for 10 min at room temperature. Finally, the cells were scanned using a Nikon A1R/A1 laser scanning confocal microscope (Nikon, Tokyo, Japan).

The indicated HEK-293T or 3D4/21 cells were seeded on 35 mm glass-bottomed cell culture dishes for 24 h, and then transfected with the indicated plasmids, in the presence or absence of the indicated drugs, or cultured under starvation conditions for indicated time. After fixing using 4% paraformaldehyde for 10 min at room temperature, the cells were then blocked and permeabilized with 5% non-fat milk with 0.2% Triton X-100 for 1 h. The cell samples were then incubated with the indicated antibodies for 2 h at 37 °C after washing three times with PBST, and with another three times of washing with PBST, the samples were then incubated with DyLight 405-, FITC-, Alexa Fluor 647-, or Alexa Fluor 546-labelled IgG antibodies for 1 h. After washing three times with PBST and stained with DAPI indicating nuclei, and the cells were observed under an N-structured illumination microscopy (SIM) Super-Resolution microscope (Nikon, Tokyo, JPN) or a Nikon A1R/A1 laser scanning confocal microscope (Nikon, Tokyo, JPN). Only cells with more than GFP dots or ring-like structures were scored as positive.

Confocal microscopy was used to observe the colocalization of HA and HSP90AA1. A549 cells were cotransfected with Flag-HA and Myc-HSP90AA1; Flag-N was used as a negative control. Twenty-four hours post-transfection, the samples were fixed in 4% paraformaldehyde for 15 min at room temperature, without permeabilized cells, were then incubated with anti-Flag and anti-Myc antibodies for 2 h at 37°C, and finally incubated with the appropriate secondary antibodies for 1 h at 37°C. Cells were stained with wheat germ agglutinin as the product instruction and were then stained with 4′,6-diamidino-2-phenylindole. After that, the cells were scanned with a Nikon A1R/A1 laser scanning confocal microscope.