Im500

Paraffin-embedded sections were used according to previously published methods (Rossi et al., 2012 (link)). Sections were incubated with a specific antibody anti-vimentin (1:1000, 137321 Abcam, Cambridge, United Kingdom). Sections were then washed with PBS and incubated with secondary antibodies. Specific labeling was detected with a biotin-conjugated goat anti-rabbit IgG and avidin-biotin peroxidase complex (1:200, BA-1000 DBA, Milan, Italy). Five distinct preparations were analyzed by a pathologist who was familiar with the protocol. A total of 23 fields of view were analyzed in each section for a total area of 4.3526e + 0.04 μm² at 400× magnification. Using computer-aided planimetry (IM500, Leica Microsystem, Milan, Italy) the percentage of positive stained area per total area analyzed was calculated. A colored threshold mask for immunostaining was defined and applied to all sections.
In addition, cardiac tissue was stained with haematoxylin and eosin and observed at a magnification of 200× and 400× using an optical microscope to evaluate morphology.

Paraffin-embedded eye samples were treated as described by Rossi et al. [12 (link)]. Eye sections were incubated with primary anti-acetyl-p53 and anti-acetyl-FOXO1 antibodies (Santa Cruz, USA) and with secondary goat anti-rabbit IgG (DBA, Milan, Italy). A negative control was performed with the primary antibody omitted (data not shown). Seven distinct tissue sections for each group of animals were done and 20 microscopic fields were analyzed in each section at 200x magnification, by an expert pathologist (intraobserver variability 6%) blinded to the experimental protocol. Of each total area, a computer-aided planimetry (IM500, Leica Microsystem, Milano, Italy) was performed and the percentage of positive stained area per total area analyzed was calculated. A color threshold mask for immunostaining was defined and applied to all sections.

We performed the SARS-CoV-2 RNA in situ hybridization (ISH) on formalin-fixed paraffin-embedded (FFPE) samples. The authors detected SARS-CoV-2 RNA using Ready-to-use reagents from RNAscope 2.5 LS Reagent Kit-BROWN and the V-nCoV2019-S probe (advanced cell diagnostics). According to the user manual, the analysis was performed on the Leica Biosystems’ BOND RX Research Advanced Staining System (Doc. No. 322100-USM). The Ubiquitin C a constitutively expressed endogenous gene was used as a positive control to assess the presence of adequate RNA quality and avoid a false negative result. The dapB test was used as a negative control to assess non-specific staining, comparing the cases with negative or weakly stained SARS-CoV-2 RNA staining. Two independent authors, blinded to the characteristics of the enrolled study population, evaluated the slides. A SARS-CoV-2 RNA ISH test result was defined as positive if the cells showed brown punctate dot-like positivity. We stained the paraffin sections with antibodies against CD31, and CD68 and quantified the percentage of positive cells for specimen area (%). We performed immunohistochemistry analysis with a personal computer-based quantitative 24-bit color image analysis system (IM500; Leica Microsystem AG).