Histology and immunohistochemistry

Paraffin-embedded lung tissue blocks for PBS-treated, Ad-Empty (2.5×10⁸ PFU) or Ad-hACE2 at doses of 2.5×10⁸-7.5×10⁷ PFU, were cut into 5μm sections. Sections were stained with haematoxylin and eosin (H&E) by the Biorepository and Pathology Core, or serial sections (5μm) provided for immunohistochemical (IHC-P) staining for hACE2 as follows; sections were deparaffinized in xylene-free clearing agent, Histo-Clear (Thermo Fisher Scientific, Waltham, MA) and rehydrated using a decreasing ethanol (EtOH) gradient. Endogenous peroxidase activity was blocked by incubating sections for 10 min in Bloxall solution (Vector Laboratories, Burlingame, CA) between the first and second 100% EtOH rehydration steps. Antigen retrieval was performed by boiling in sodium citrate (pH 6.0) solution, as described previously [32]. Slides were allowed to cool and were washed with PBS prior to blocking of endogenous biotin or avidin binding proteins, using the Avidin/Biotin Blocking Kit (Vector Laboratories, Burlingame, CA) as recommended by the manufacturer. Sections were blocked for 30 min at 37˚C with blocking buffer provided in the VECTASTAIN® Elite ABC-HRP Kit (Rabbit IgG) with the addition of 1% (w/v) BSA to act as a stabilizer and 0.1% (v/v) Triton X-100 to facilitate permeabilization of the tissue. Following blocking, tissue sections were separated using a hydrophobic pen. A monoclonal rabbit isotype control [clone #SP137; Abcam, Cambridge, MA] or monoclonal rabbit anti-human ACE2 antibody (clone #EPR4436; Abcam Cambridge, MA) diluted in blocking buffer (see above) were added to one section on each slide at a final concentration of 1.33μg/mL for 1 h at room temperature. Following incubation, sections were washed three times in PBS. Biotinylated anti-rabbit secondary antibody was prepared as instructed by guidelines for the VECTASTAIN® Elite ABC-HRP (Rabbit IgG) kit and sections incubated for 30 min at room temperature. Following incubation, sections were washed in PBS and the VECTASTAIN ELITE ABC reagent was prepared and allowed to stand at room temperature for 30 min. The pre-made Elite ABC reagent was then added to sections for 30 min at room temperature. Cells were washed again three times in PBS and the 3,3’-diaminobenzidine (DAB) chromogen DAB Peroxidase (HRP) Substrate Kit (Vector Laboratories, Burlingame, CA) prepared immediately prior to use and addition to each slide individually. Development of a positive control slide was timed under a microscope and the same development time (10 min) applied to all other sections. Sections were transferred to distilled H₂O and nuclei counterstained for 45 s using Haematoxylin QS (Vector Laboratories, Burlingame, CA) before being rinsed extensively with tap water. Sections were then dehydrated by using an increasing gradient of EtOH, ending in Histo-Clear solution.

Lung and brain tissue from C57BL/6J mice pre-treated with 2.5×10⁸ PFU Ad-hACE2, or K18 transgenic mice, was harvested at D0, D2, D5 and D6 post-infection with 1×10⁴ PFU of SARS-CoV-2. Tissue was fixed and processed for H&E staining as described above. IHC-P staining for SARS-CoV nucleocapsid (N) protein was largely performed as above, with some modifications: endogenous peroxidase activity was blocked using Bloxall solution following rehydration and antigen retrieval, serum blocking was performed using 4% (v/v) normal goat serum (Vector Laboratories, Burlingame, CA) and the Avidin/Biotin blocking step was carried out immediately prior to primary antibody staining. 5μm lung or brain sections were incubated overnight at 4°C with polyclonal IgG control (Abcam, Cambridge, MA), or polyclonal anti-SARS-CoV N antibody (Novus Biologicals, cat #NB100-56576), both at a final concentration of 2 μg/mL. Subsequent steps were performed as described for hACE2 IHC-P, with a nova red HRP substrate used for development (Vector® NovaRED® Substrate Kit, Peroxidase) instead of DAB. Sections were mounted using Histomount Solution (Life Technologies) and provided to a veterinary pathologist at the Center for Comparative Medicine and Surgery (CCMS) Comparative Pathology Laboratory, ISMMS (Dr. Virginia Gillespie). The pathologist evaluated and photographed IHC-P for hACE2, IHC-P for SARS-CoV N, and H&E sections, and was blinded to the treatment groups. Images were captured under 20X, 100X or 200X magnification using an Olympus BX43 with the Olympus DP21 Digital Camera system. When possible, representative images from each group are shown alongside a matched monoclonal or polyclonal IgG control antibody, and H&E section from the same region of tissue. Lung H&Es were evaluated using a pathological scoring system to assess nine parameters: amount of lung affected, perivascular inflammation, epithelial degeneration/necrosis of bronchi/bronchioles, bronchial/bronchiolar inflammation and intraluminal debris in bronchi/bronchioles, as well as alveolar inflammation, necrosis in alveoli, fibrin deposition and Type 2 pneumocyte hyperplasia. For area affected a ranking of 0–4 was used where 0 = not affected, 1 = 25%, 2 = 25-50%, 3 = 50-75% and 4 = 100% of lung affected was used. For histological parameters a score of 1 indicated mild, 2 = moderate, 3 = marked and 4 = severe.