2.2. Immunohistochemistry and ACE2 Scoring

All tissue samples were fixed in 4% buffered formaldehyde for 24 h, dehydrated and embedded in paraffin. Immunohistochemistry was performed on 5-µm-thick sections mounted on silane-coated glass slides. After deparaffinization with xylene and rehydration with decreasing concentrations of ethanol, sections were incubated with 4.5% hydrogen peroxide in distilled water for 10 min to block endogenous peroxidase activity and rinsed in distilled water. Tissues were then heated in a pressure cooker for 6 min to induced antigen retrieval in a 0.01 M citrate buffer solution (pH 6.2) (Scytek, West Logan, UT, USA). After thorough washing with phosphate-buffered saline (PBS), the sections were incubated for 30 min in a solution of 0.5% casein in PBS and exposed to ACE2 primary antibodies. The polyclonal rabbit anti-ACE2 (ab15348, Abcam, Cambridge, UK) and the monoclonal mouse anti-ACE2 (MAB933, R&D systems, Minneapolis, MN, USA) were diluted (1:1500 and 1:50, respectively) in PBS with 0.05% casein, were applied on sections and incubated overnight at 4 °C. Subsequently, the samples were incubated with a PowerVision Poly HRP-anti-rabbit and anti-mouse IgG (Klinipath, Duiven, Holland), and the antigens were visualized via the addition of a solution of 3-3′-diaminobenzidine tetrachloride (DAB)—H₂O₂ buffer (Liquid DAB, San Ramon, CA, USA) before being counterstained with Mayer’s hemalum (Klinipath, Duiven, Holland) and Luxol fast blue and finally mounted with a synthetic balm (Thermo Scientific, Pittsburg, PA, USA). To exclude antigen-independent staining, negative controls for which the incubation step with the primary antibody was omitted were examined. In all instance, these controls did not reveal any staining. ACE2 immunoreactivity was initially detected in positive controls (kidney and small intestine tissues) known to express ACE2 (The Human Protein Atlas) (Figure 1).

Negative and positive controls for ACE2 used for both antibodies. Kidney tissues were used as negative controls and kidney or small intestine tissues for positive controls. Scale bar = 100 µm.

The slides were analyzed using a BX43 Olympus microscope. The percentage of stained cells and staining intensity were semi-quantitatively assessed by one pathologist (LV) using a 4-grade scale for both parameters. The percentage of positivity was described as 0 = no stained cells, 1 = less than 25%, 2 = between 25 to 75%, 3 = above 75%. The intensity of the staining is counted as 0 = no expression/intensity, 1 = weak intensity, 2 = moderate, 3 = strong). Positivity in inflammatory cells and in fibroblasts were assessed as present or absent. An immunostaining score was finally calculated by the sum of these two values (percentage + intensity) and a value from 0 to 6 was attributed to each cell type, and tissue location analyzed.