Cellsens software v 1.18

Paraffin embedded tissue sections were stained for SARS-CoV-2 nucleocapsid protein (1:500), microtubule-associated protein 2 (Abcam; ab32454; 1:500), ionized calcium binding adaptor molecule 1 (Abcam; ab5076; 1:50)/glial fibrillary acidic protein (Sigma; 3893;1:500) using a Leica Bond RXm automated staining instrument following permeabilization using 0.01% Triton X diluted in Tris-buffered saline (TBS). Blocking was performed with 1% donkey serum diluted in TBS. Sections were stained for DAPI (Sigma) and mounted on glass coverslips in ProLong Gold Antifade mounting medium and stored at ambient temperature until imaging. Images were captured using an Olympus BX63 fluorescence microscope equipped with a motorized stage and Hamamatsu ORCA-flash 4.0 LT CCD camera. Images were collected and regions of interest quantified with Olympus cellSens software (v 1.18) using an Olympus X-line apochromat 10X (0.40 N.A.), 20X (0.8 N.A.) or 40X (0.95 N.A.) air objectives, or Uplan Flour X100 oil immersion (1.3 N.A.) objective.

Paraffin embedded tissue sections were stained for SARS-CoV-2 nucleocapsid protein (1:500) and ionized calcium binding adaptor molecule 1 (Abcam, ab5076; 1:50) using a Leica Bond RX^m automated staining instrument following permeabilization using 0.01% Triton X diluted in Tris-buffered saline (TBS). Blocking was performed with 1% donkey serum diluted in TBS. Sections were stained with DAPI (Sigma) and mounted on glass coverslips in ProLong Gold Antifade mounting medium and stored at ambient temperature until imaging. Images were captured using an Olympus BX63 fluorescence microscope equipped with a motorized stage and Hamamatsu ORCA-flash 4.0 LT CCD camera. Images were collected with Olympus cellSens software (v 1.18) using an Olympus X-line apochromat 10× (0.40 N.A.), 20× (0.8 N.A.) or 40× (0.95 N.A.) air objectives, or Uplan Fluor ×100 oil immersion (1.3 N.A.) objective. Regions of interest (ROIs) were drawn around the perimeter of each tissue section and intensity thresholds were kept constant across groups analyses were performed blinded. Co-localization and intensity measurements were obtained using the Count and Measure feature of cellSens.

The analysis of the wound measurements was carried out using Image ProPlus software V7 (MediaCybernetics Inc., Rockville, MD, USA; https://www.meyerinst.com/mediacybernetics/image-pro-plus/, accessed on 12 January 2022). Cell counts and intensity measurements were carried out using CellSens software V1.18 (Olympus, Australia; https://www.olympus-lifescience.com/en/software/cellsens, accessed on 29 June 2022). Statistical differences were determined using ANOVA, with the Bonferroni correction for multiple groups and Student’s t-test. For non-parametric data, a Mann–Whitney U test was used. A p value of <0.05 was considered significant.