Zen light edition software

Cells seeded on coverslips were incubated with AFB1 (40 μm) at 37 °C for the indicated times. The cells were fixed in 4% paraformaldehyde for 20 min and permeabilized with 0.1% Triton X-100 at room temperature. The cells were then blocked in 5% bovine serum albumin and incubated with the Cav-1 antibody at a dilution of 1:1000 at room temperature for 2 h. After three washes with PBS, the cells were incubated with Alexa Fluor 488-conjugated secondary antibodies for 1 h. The cells were washed three times again with PBS and the nuclei were stained with 4′,6-diamidino-2-phenyl-indole (Roche) for 10 min. Images were obtained using a confocal laser scanning microscope (Zeiss LSM710 Meta, Carl Zeiss). The fluorescence intensity of the images was processed and quantified using ZEN Light Edition software (Carl Zeiss).
For autophagic marker detection, cells seeded on coverslips were washed with culture medium and then incubated at 37 °C for 30 min with 250 μl of 1 μmol/l DALGreen working solution. After a 6 h incubation with fresh culture medium, the cells were washed with Hanks’ HEPES buffer twice and DALGreen was observed by a confocal laser scanning microscope. The images were processed and analyzed using ZEN Light Edition software.

For histological analyses, lungs were inflated with 4% paraformaldehyde or formalin (3.7% formaldehyde) and fixed for 36 h. Fixed paraffin sections were rehydrated, subjected to antigen retrieval, blocked in TBS (0.1% Triton X-100 containing 1% BSA) or DAKO protein-free blocking solution, and sequentially incubated with specific primary antibodies, biotinylated secondary antibodies (DAKO), and the Alexa Fluor system (Invitrogen, Waltham, MA, USA). Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining was performed using the in situ Cell Death Detection kit (Roche). Images were produced using a conventional microscope mounted with a DP71 digital camera (Olympus), an LSM 710 T-PMT confocal microscope (Carl Zeiss), and an AXIO Zoom.V16 and ApoTome.2 (Carl Zeiss). Images were processed with equivalent parameters using the ZEN Light Edition software (Carl Zeiss) https://www.zeiss.com.cn/microscopy/products/microscope-software/zen-lite/zen-lite-download.html accessed on 6 October 2023.

Cells were seeded in four-well plastic dishes to measure cell proliferation. For each condition, cells were washed twice with PBS and fixed with 4% formaldehyde in 0.5 ml PBS for 20 min at room temperature. Then, the cells were washed again with PBS. Next, the cells were blocked in PBS containing 0.5% BSA and incubated for 1 h with polyclonal Ki-67 antibody (Millipore). Then, the cells were washed three times for 10 min with PBS and incubated with FITC-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch, West Grove, PA, USA) secondary antibody for 1 h. All images were visualized by confocal microscopy (LSM 710; Zeiss), and the acquired images were transferred to a computer equipped with Zen Light Edition software (Zeiss).

Cells seeded on coverslips were incubated with virus at 4 °C for 1 h, and then virus entry was initiated by moving the cells to 37 °C. At the indicated time points, the cells were fixed in 4% paraformaldehyde for 20 min and permeabilized with 0.1% Triton X-100 at RT. The cells were then blocked in 5% BSA and incubated with an Alexa Fluor 488 (AF 488)-conjugated anti-mouse antibody (Invitrogen) and Alexa Fluor 555 (AF 555)-conjugated anti-rabbit antibody (Invitrogen) for 2 h. After three washes with PBS, the cells were incubated with fluorochrome-conjugated secondary antibodies for 1 h. The cells were washed again three times with PBS, and the nuclei were stained with 4′,6-diamidino-2-phenyl-indole (DAPI, Roche) for 10 min. Images were obtained using a confocal laser scanning microscope (Zeiss LSM710 Meta; Carl Zeiss). The fluorescence intensity of the images was processed and quantified using ZEN Light Edition software (Carl Zeiss).