Fv10 asw viewer software

Cells grown on coverslips were fixed in 4% paraformaldehyde. Fixed cells were permeabilized with 1% Triton‐100 and blocked with 10% normal goat serum. Cells were then incubated with primary antibody for 2 h at 37° in a humid chamber and were than incubated with the secondary antibodies for 1 h. Immunofluorescence images were captured using with FV10‐ASW viewer software (Olympus, Tokyo, Japan). Primary antibodies used were γH2AX (Cell Signaling, Danvers, MA), FUNDC1, and LC3 (Abcam, Cambridge, MA).

Cells were grown on coverslips for 24 h and fixed in 4% paraformaldehyde at room temperature and permeabilized with 0.1% Triton X-100. Cells were incubated with 5% bovine serum albumin (BSA) solution at 37°C for 30 min to block nonspecific interactions. Cells were incubated serially with anti-αSMA and anti-FAPα primary antibodies (abcam, ab32575, ab53066), at 4°C overnight, and then with secondary antibodies (Beyotime, P0176-1) at room temperature in the dark for 1 h. Cells were co-stained with DAPI (4′,6-diamidino-2-phenylindole) to detect nuclei. Immunofluorescence images were captured using FV10-ASW viewer software (Olympus, Tokyo, Japan).

To identify the cell subsets expressing PD-L1 in the TME, multiplex immunofluorescence staining was obtained using TSA Plus Fluorescence Kits (PerkinElmer, Foster City, CA, USA) combined with IHC (TSA-IHC). Different primary antibodies were sequentially applied, followed by horseradish peroxidase-conjugated secondary antibody incubation and tyramide signal amplification. The slides were microwave heat-treated after each TSA operation. Nuclei were stained with 4′-6′-diamidino-2-phenylindole (DAPI, Thermo Scientific) after all the human antigens had been labelled.
To obtain multispectral images, the stained slides were scanned using the Vectra System (PerkinElmer), which captures the fluorescent spectra at 20-nm wavelength intervals from 420 to 720 nm with identical exposure time; the scans were combined to build a single stack image.
For colocalisation analysis, images were acquired using a laser confocal microscope (Olympus, Essex, UK) and analysed using FV10-ASW Viewer software (Olympus).

Cells were grown on glass coverslips up to 80% confluency. Then, cells were washed twice with PBS, fixed in 4% paraformaldehyde and processed for immunofluorescence staining. Frozen tissues were embedded in OCT and sectioned, then mounted on glass slides. The mounted tissues were air dried overnight fixed in 4% paraformaldehyde. The cell climbing slices and frozen tissue section were double immunostained with mouse anti-human antibody phospho-Histone H2AX (γ-H2AX) and rabbit anti-human 53BP1 antibody in a humidified chamber overnight at 4 °C. Cells were washed with PBS twice and primary antibodies were visualized by DyLight 549 conjugated Goat anti-Mouse IgG and Alexa Fluor 488 conjugated Goat anti-Rabbit IgG. DNA were stained with DAPI. Images were collected on the Olympus FluoView confocal microscopes and analyzed with FV10-ASW viewer software (Olympus, Tokyo, Japan). The number of 53BP1 and γ-H2AX fusion co-positive (yellow) foci per nucleus was calculated according to the formula ${\sum }_{\text{foci}}/{\text{n}}_{\text{total}\text{cells}}.$ The ${\sum }_{\text{foci}}$ means all the foci recorded in the cells counted at a given time point and the n_{total cells} represents the total number of cells assessed at that time point. All counting was performed manually by a single operator to minimize variation and then statistical analyzed. All experiments were repeated at least three times.