Vector antigen unmasking solution

Animal lung tissues were collected and fixed with 10% formalin for 24 h and paraffin-embedded. Three sections from each animal were used for histology analysis. Upon staining, sections were mounted onto slides and dewaxed in xylene. Antigen retrieval was performed by autoclaving slides at 121 °C for 3 min in Vector antigen unmasking solution, citrate-based (Vector Laboratories, Inc., Newark, CA, USA). Sections were then permeabilized with 0.1% Triton X-100, quenched by Sudan Black B, and blocked with 1% BSA. Infected cells were stained by in-house rabbit anti-SARS-CoV-2 nucleoprotein polyclonal antibody and detected by anti-rabbit Alexaflour 488 (Abcam). Cell nuclei were stained with Hoechst 33258 (Thermo). For H&E staining, tissue sections were stained with Gill’s haematoxylin and eosin-Y. Images were acquired using the Olympus BX53 light microscope (EVIDENT, Tokyo, Japan).

Human striated skeletal muscle (piloerector muscle) in skin samples were obtained after informed consent from patients attending the Vancouver General Hospital Skin Care Center, University of British Columbia, Vancouver, British Columbia, Canada, and performed in accordance with the ethical principles set for in the Declaration of Helsinki and at the University of British Columbia. Samples were fixed in 10% neutral buffered formalin and embedded in paraffin.
Sections (5 μm thick) were then deparaffinized in xylene and rehydrated in graded alcohols. For Panx1, antigen retrieval was done using Vector Antigen Unmasking Solution (Vector Laboratories, Burlingame, CA) according to the manufacturer's instructions. Antigen retrieval using sodium citrate buffer was used for Panx3 (3 (link), 7 (link)). Tissue sections were labeled with anti-human Panx1 (1:50) or anti-Panx3 (CT-379, 1:50) (3 (link), 7 (link)). Appropriate secondary antibodies conjugated to Texas-Red (Jackson ImmunoResearch, West Grove, PA, 1:100) were applied. A peptide pre-adsorption assay was done to verify antibody specificity as previously described (3 (link), 7 (link)). Hoechst 33342 (Molecular Probes, Eugene, OR) was used as a nuclear stain, and labeling was visualized with a Zeiss LSM 510 META inverted confocal microscope.

After euthanasia, wounds with surrounding tissues were excised and fixed with 10% phosphate-buffered (pH = 7.4) formalin. The samples were transversely cut exactly through the center of the wound, dehydrated, and paraffin-embedded according to the routine protocols. Cross sections (5 μm thick) were stained with hematoxylin and eosin (H&E) and Mallory's trichrome stain. For immunostaining, sections were treated with 3% H₂O₂ for 10 min and then with 10% nonimmune goat serum before incubation with rabbit polyclonal antibodies against α-smooth muscle actin (α-SMA) and CD31 (Abcam, UK) or rat monoclonal antibodies against f4/80 (Serotec, UK). Goat anti-rabbit and anti-rat IgG biotinylated antibodies (Vector, USA) were applied and stained with avidin-peroxidase conjugate and diaminobenzidine (Vector, USA). Before CD31 staining, heat-mediated antigen retrieval with Vector Antigen Unmasking Solution (Vector, USA) at 98°C for 40 min was applied additionally.
Samples were analyzed with a DM 5000B microscope equipped with a DFC 320 digital camera (Leica, Germany).

Human-derived hippocampal and temporal lobe sections and mice-derived slices embedded in paraffin were immunostained after a dewaxing procedure comprising sequential washing steps with xylene, ethanol and PBS. Sections were immunostained with S1T [13 (link)] or Iba1 antibodies after antigen unmasking using a Vector Antigen unmasking solution (Vector Laboratories). For 6E10 staining, sections were treated with formic acid (90% for 5 min). Serial slices were immunostained with S1T and 6E10 antibodies. Non-specific binding was blocked for 1 h in 5% BSA, 0.5% TBS-Triton X-100 solution. Sections were incubated at 4 °C overnight with primary antibodies (Supplementary Table S3). Sections were incubated with HRP-conjugated secondary antibodies (Jackson ImmunoResearch) followed by diaminobenzidine (DAB) substrate (Vector). Neuronal nuclei were detected with Cresyl violet.

Antigen retrieval was carried out by placing free-floating sections in Vector Antigen Unmasking solution (1:100 in 0.1 M PB; Vector Labs, Burlingame, CA, United States) heated to 80 degrees °C for 1 h. For dual labeling, sections were co-incubated in primary antibodies (NG2, 2 μl:1000 μl, BCAN, 2 μl:1000 μl; SMI-312, 0.5 μl:1000 μl) in 2% bovine serum albumin (BSA) for 72 h at 4°C. This step was followed by 4 h incubation at room temperature in Alexa Fluor goat anti-mouse 594 (1:300 μl; A-11005, Invitrogen, Grand Island, NY, United States) and donkey anti-rabbit 488 (1:300 μl; A-21206, Invitrogen, Grand Island, NY, United States), followed by 10 min in 1 mM CuSO4 solution (pH 5.0) to block endogenous lipofuscin autofluorescence (Schnell et al., 1999 (link)). Sections were mounted and coverslipped using Dako mounting media (S3023, Dako, North America, Carpinteria, CA, United States).