Histopathology and immunohistochemistry

Formalin-fixed samples were routinely processed and embedded in paraffin wax (Surgipath Paraplast, Leica Biosystems). Formalin-fixed paraffin-embedded tissue blocks were sectioned at 4-μm-thick sections using a standard rotary microtome (HM-340E, Thermo Fisher). Tissue sections were stained with hematoxylin and eosin.

Abnormalities in the tracheal and pulmonary tissue were scored using representative microscopic lesions. Scoring criteria ranged from 0 to 3 based on the severity or proportion of the lesioned tissue: 0, none-to-rare or <10%; 1, mild or 10%–40%; 2, moderate or 40%–70%; and 3, severe or >70%. Tracheal abnormalities were scored using the following criteria: (1) inflammation of lamina propria; (2) cellular exudates in tracheal lumen; and (3) tracheal epithelium damage. Lung abnormalities were scored using the following criteria: (1) inflammation of the peribronchiolar region; (2) inflammation of the perivascular region; (3) cellular exudates in bronchiolar lumen; (4) bronchiolar epithelium damage; (5) thickening of the alveolar wall (interstitial pneumonia); and (6) hemorrhage. While scoring, care was taken because lesions were not evenly distributed across the entire tissue and showed various patterns. The final score was the sum of scores under each criterion; a high score indicated a higher degree of microscopic damage. The criteria used for scoring are categorized and summarized in Table S1.

For immunohistochemistry, a silane-coated slide was used for its strong adhesiveness. To re-establish immunoactivity, antigen retrieval was conducted using citrate buffer (pH 6.0) at 95°C for 30 min and at room temperature for 20 min. Sections were then incubated overnight at 4°C with a SARS-CoV-2 NP antibody (40,143-V08B, Sino Biological) or Ly6G (14-5931-82, Thermo Fisher), and F4/80 (SC-25830, Santa Cruz Biotechnology) diluted to 1:500 using an antibody diluent (E09-300, GBI Labs). To label SARS-CoV-2 NP, Ly6G, and F4/80, horseradish peroxidase-conjugated anti-rabbit and anti-rat immunoglobulin G (IgG) antibodies (MP-7500 and MP-7404, Vector Laboratories) were used. The antibody was visualized with 3,3′-diaminobenzidine (SK-4105, Vector Laboratories) at the concentration recommended by the manufacturers. Tissues were counterstained with methyl green. All of the slides were examined (BX53, Olympus) and imaged (DP80, Olympus) microscopically using a light microscope. All histopathologic examinations were conducted in a double-blind fashion with trained pathologists. To quantify immunohistochemical results, image analysis was performed using TS Auto 5.1 (Olympus). The percentage of immunopositive area was analyzed at a defined magnification and area (400 magnification field, 0.144 mm²).

For immunofluorescence staining, we used the method of intratracheal instillation of the fixative. Briefly, tracheostomy was performed, and an 18G needle tip was inserted into the trachea. Lungs were inflated via cannula by gentle infusion of 10% neutral buffered formalin at a constant fluid pressure of 25 cm for 20 min. After tying off the trachea, the lungs were kept in a 50-mL tube containing 10% neutral buffered formalin (NBF) for 6 h, and then, transferred to a new tube containing pre-chilled cryoprotection solution (20% sucrose and 33% OCT [optimal cutting temperature] compound) and kept overnight. The following day, the lungs were embedded in OCT compound and snap-frozen. Cryosections (8-μm-thick) were prepared using a cryostat. For staining, cryosections were blocked with 5% normal donkey serum in PBS containing 0.3% Triton X-100 (PBS-T) and incubated overnight at 4°C with the following antibodies in PBS-T: rabbit anti-AQP5 (#ab78486, Abcam), rabbit anti-proSPC (#AB3786, Sigma-Aldrich), rabbit anti-VWF (#PA5-16634, Thermo Fisher), rabbit anti-ACE2 (#A12737, ABclonal), chicken anti-GFP (#ab13970, Abcam). The next day, the sections were washed with PBS for 2 h and incubated for 90 min with the following secondary antibodies in PBS containing 0.1% Triton X-100 and 0.1% Tween 20: donkey anti-rabbit (#{"type":"entrez-protein","attrs":{"text":"A31573","term_id":"87384","term_text":"pir||A31573"}}A31573, Thermo Fisher) or donkey anti-chicken (#703-545-155, Jackson ImmunoResearch). After washes with PBS for 90 min, samples were mounted with ProLong Gold containing DAPI ({"type":"entrez-protein","attrs":{"text":"P36941","term_id":"549090","term_text":"P36941"}}P36941, Thermo Fisher), and images were acquired using a confocal microscope (LSM 900, Carl Zeiss).