Tmr red

Cells grown in monolayer cultures were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS), permeabilized with 0.2% Triton X-100 and blocked with 10% goat serum prior to antibody staining. Specific primary antibodies were added at 1:100 dilution and incubated at 4 °C overnight. Fluorescent staining was developed using the FITC or Cy3 fluorescence system (Sigma-Aldrich, St. Louis, MO, USA). TUNEL assay, in situ cell death detection kit (TMR red; Boehringer-Mannheim, Mannheim, Germany) was used. The sections were incubated with the TUNEL reaction solution for 60 min at 37 °C in the dark. Cover slips were mounted onto slides with Vectashield mounting medium with DAPI (H-1200; Vector Laboratories Inc.). Fluorescent images were collected by using a Zeiss laser scanning microscope (LSM) 510 confocal microscope, and images were captured with LSM software, version 2.3 (Carl Zeiss, Wetzlar, Germany). Apoptotic cells and total cells were counted in ten randomly chosen microscopic fields, and the percentage of apoptotic cells was calculated and compared between the experimental and control groups.

Proliferative beta-cells were identified by insulin-Ki67 double immunofluorescence staining with rabbit anti-Ki67 (1:200; Thermo Fisher), guinea pig anti-insulin antibody (1:100; Abcam) and relevant secondary antibodies (1:1000; Abcam). Apoptotic beta-cells were identified by insulin and terminal deoxynucleotidyl transferase dUTP nick end labeling (Tunel) (TMR red, Roche, Mississauga, ON, Canada) dual labeling. Results are expressed as the percentage of Ki67+, or Tunel+ beta-cells.

Lungs were collected from indicated mice sacrificed on day 6 post-infection. Tissues were covered in cryo-embedding media, kept in liquid nitrogen until completely submerged, then stored at −80 °C until ready for sectioning. 4 mm sections were cut with a cryotome and stained with haematoxylin and eosin. For immunofluorescence, sections were fixed in 4% paraformaldehyde for 1 h at RT, washed with PBS, then incubated in “permeabilisation solution” containing 0.05% TX-100 and 0.1% sodium citrate for 2 min on ice (4 °C). Cell death was analyzed with an in situ cell death detection kit (TMR-red, Roche) after antigen retrieval either alone or followed by staining with anti-CD45 (AB 10558 Abcam) and Goat anti-Rabbit IgG DyLight488 (ThermoFicher ref: 35553). Anti-CD8a (AB 217344, Abcam) was used 1/100 combined with a secondary Goat anti-Rabbit IgG AF568. Hoechst 33342 was used to stain nuclei. Brightfield and fluorescence microscopy was performed using ZEISS Axio Scan.Z1. Samples were analyzed with Zen 3.2. blue edition (Zeiss) and quantified with QuPath software 0.2.3.

All the enzyme-linked immunosorbent assays for the study were performed according to manufacturer’s instructions. The In Situ Cell Death Detection Kit TMR red has been utilized for the paraffin-embedded (3 µm) thick rat lung sections following the recommendations by the company (12156792910, Roche, Basel, Switzerland). Recombinant proteins and ELISA kits used for the study are represented in Supplementary Table 3.

To detect apoptosis, the terminal deoxynucleotidyl transferase (TdT) nick end labelling test by the In Situ Cell Death detection kit, TMR red (Roche, Mannheim, Germany) was employed as instructed by the manufacturer and described earlier [40 (link)].