Histopathology Scoring and SARS-CoV-2 Antigen Detection

Haematoxylin and Eosin (H&E) staining of formalin-fixed and paraffin-embedding tissue sections (4 μm each) were observed for histopathology changes. Severity of histopathology in lungs were given score under complete masking²⁷ (link) by assessment of pulmonary congestion, interstitial infiltration, alveolar infiltration, hemorrhage and scored 0–4 as described previously.²⁶ The following criteria were used for scoring: 0, normal lung section; 1, blood vessel congestion, perivascular or peribronchiolar infiltration; 2, in addition to 1 with diffuse alveolar wall congestion and infiltration; 3, air space infiltration, exudation, hemorrhage of localized alveolitis; 4, diffuse alveolitis were observed. Immunohistochemistry staining was performed by a DAB (3,3′-diaminobenzidine) substrate kit (Vector Laboratories) as we previously described.²⁸ Briefly, For ACE2 antigen detection, ACE2 recombinant rabbit monoclonal antibody (MA5-32307, Invitrogen) were used and followed with color development by using the DAB substrate kit. The ACE2 protein was detected by haematoxylin and then mounted the tissue sections with the VectaMount permanent mounting medium (Vector Laboratories). For SARS-CoV-2 antigen expression, slides of lung and NT tissues were stained with an in-house antibody of rabbit anti SARS-CoV-2 nucleocapsid protein (NP) followed by a secondary antibody of FITC–conjugated goat anti rabbit IgG (65-6111, Thermo Fisher Scientific, Waltham, MA, USA). The following criteria were used for NP scoring. Lung: “score 0”- no fluorescence staining signal; “score 1”- only in 1–3 bronchiolar epithelium with N antigen positive cells; “score 2”- more than 3 bronchiolar epithelium with N antigen positive cells; “score 3”- Bronchiolar epithelium with a few positive cells in nearby alveolar; “score 4”- multiple foci or large area of alveoli with N antigen positive cells. NT: “score 0”- no fluorescence staining signal; “score 1”- a few N antigen positive cells scattered in the epithelium; “score 2”- epithelium showing continually positive N antigen focus in adjacent cells; “score 3”- more N antigen positive of epithelial foci distributed in different area. Images were captured by using microscope of Olympus BX53 semi-motorized fluorescence or bright-field with OLYMPUS CellSense Standard Software.