Axioimager z2 system

The lungs from SARS-CoV-2 infected and control mice were inflated and fixed in 10% formalin for seven days. Lungs were embedded in paraffin and sectioned before hematoxylin and eosin staining. Lung slides were scanned using the Hamamatsu NanoZoomer slide scanning system and head sections were imaged using the Zeiss AxioImager Z2 system.

The lungs and heads from SARS-CoV-2 infected and control hamsters were fixed in 10% formalin for seven days. Lungs were embedded in paraffin and sectioned before hematoxylin and eosin staining (H & E) and RNA in situ hybridization (RNA-ISH) to detect SARS-CoV-2 RNA. Following formalin fixation, heads were decalcified in 0.5 M EDTA for seven days, cryoprotected in three exchanges of 30% sucrose for three days, and then embedded in O.C.T. compound before RNA-ISH. RNA-ISH was performed using a probe against the S gene of SARS-CoV-2 (V-nCoV2019-S, Cat #848561) with the RNAscope® 2.5 HD Assay—BROWN (ACDBio, Cat#322310) according to the manufacturers’ recommendations. Lung slides were scanned using the Hamamatsu NanoZoomer slide scanning system and head sections were imaged using the Zeiss AxioImager Z2 system. Lung sections were scored according to a previous publication (Imai et al., 2020 (link)) (< 10% affected lung tissue = 1, > 10% but < 50% affected area = 2, > 50% affected area = 3).

Organ samples were fixed in fresh 4% paraformaldehyde at 4 °C overnight and further subjected to routine histological procedures for embedding in paraffin. 5-μm sections of the samples from at least three different animals per group were placed on microscopic slides next to one another to enable cross-comparison within a slide after H&E staining or IHC staining with the antibodies indicated. Antibodies used were GFP (D5.1) XP, PTEN (D4.3), phospho (p)-AKT (S473) (D9E), p-ribosomal protein S6 (S235/236) (D57.2.2E), Cleaved Caspase-3 (Asp175) (all: Cell Signaling Technologies, Danvers, MA), Ki67 (VP-K451, Vector Laboratories, Burlingame, CA), and CD3 (A0452) (DAKO, Glostrup, Denmark). Stained slides were scanned with a Pannoramic Scan 250 Flash or MIDI system and images acquired using Pannoramic Viewer 1.15.2 (both: 3DHistech, Budapest, Hungary). Additional images were taken on a Zeiss Axio Imager.Z2 system.

One lobe of the lung was harvested from mice at 0 or 4 days p.i. and fixed with 4% PFA–PBS for a minimum of 3 days. Formalin-fixed paraffin-embedded lung tissues were deparaffinized through sequential washes twice each in xylene and 100% ethanol for 5 min. Tissues were then pretreated with RNAscope hydrogen peroxide for 10 min at room temperature (RT) and then with RNAscope target retrieval for 5 min at 95°C to 100°C, followed by RNAscope protease plus for 30 min at 40°C. RNA-ISH was performed using a probe against the S gene of SARS-CoV-2 (V-nCoV-2019-S; ACD) using the RNAscope 2.5 HD assay—brown according to the manufacturer’s instructions. Slides were coverslipped with ProLong gold antifade mountant (Thermo Fisher). Images were acquired using a Zeiss AxioImager Z2 system with Zeiss software.