Aperio at2 system

Treated Alpl^Prx1/Prx1 and Phospho1^−/− mice were anesthetized with an intraperitoneal administration of Avertin and euthanized by exsanguination after 60 and 90 days, respectively. Skeletal/dental tissues were collected, fixed in 4% paraformaldehyde/PBS or Bouin's solution, and processed for histological analysis. The long bones and vertebrae were placed in 0.125 M EDTA/10% formalin (pH 7.3) solution for 7 days for decalcification and then were paraffin‐embedded and sectioned at 5 μm thickness. Hemimandibles were fixed in Bouin's solution, decalcified in an acetic acid/formalin/sodium chloride solution, processed for paraffin embedding, and sectioned at 6 μm thickness. Hematoxylin and eosin (H&E) and alizarin red staining were performed according to standard methods. Aperio AT2 system (Leica Biosystems of Leica Microsystems Inc., Buffalo Grove, IL, USA) was used to scan the slides. The quantification of alizarin red staining was done by Aperio Color Deconvolution Algorithm Software (Leica Biosystems). Calcified sites are shown as a percentage of strong positive alizarin red staining.

FFPE specimens were sectioned at 5 μm onto charged slides (Fisher Scientific, Cat # 22-042-924). Slides were dried for 1 h at 60 °C, deparaffinized in xylene, rehydrated through a graded series of ethanols, and rinsed in distilled water. Slides were hematoxylin (Richard-Allan Scientific, Cat# 7211) and eosin (Leica, Cat# 3801619) stained using standard laboratory protocol³⁹ . Upon completion of staining, slides were dehydrated through a series of ethanols and xylene, and mounted with Cytoseal 60 (Richard-Allan Scientific, Cat# 8310-4). Slides were scanned using a Leica Biosystems Aperio AT2 System and digitally archived via eSlide Manager (Version 12.3.2.5030). Following scanning, H&E slides were immersed in xylene to remove coverslips. Slides were rehydrated through xylene, graded ethanol, and running distilled water, decolorized with 10% acetic acid in 70% ethanol for 1 h, rinsed in distilled water, and then further decolorized in 70% ethanol for 2–3 h. Evaluation for the decolorization end-point was checked every 30 min^{40 (link),41} . Once decolorization was complete, slides were rinsed in running distilled water.

Paraffin-embedded tissue samples were cut into 5 µm sections, deparaffinized and rehydrated. For antigen retrieval, slides were boiled in citrate buffer (0.01 M, pH 6.0) using a microwave oven on high power for 5 min and cooled down to room temperature. After incubation in 3% aqueous H₂O₂ to quench the endogenous peroxidase, the sections were washed in PBST (PBS with 0.1% Tween-20, v/v) washing buffer, blocked with 5% goat serum (EMD Millipore) diluted in PBST at room temperature for 1 h and incubated with primary antibodies diluted in 2% goat serum in PBST at 4 °C overnight.
For immunohistochemistry, slides were incubated with appropriate biotinylated secondary antibodies (Supplementary Table 3) for 1 h, and then processed according to the ABC Peroxidase Standard Staining Kit (Thermo Fisher Scientific) for 30 min. The slides were stained with 3,3′-diaminobenzidine (Abcam) for 5 s to 5 min and counterstained with haematoxylin (Thermo Fisher Scientific) for 45 s. The images were scanned using the Leica Aperio AT2 system at Stanford Human Pathology/Histology Service Center. Serial sections were incubated with GS and MYC-tag primary antibodies, and a cluster of cells (at least 20 cells) positive both for GS and MYC-tag were counted as early foci. The antibodies used in this study are shown in Supplementary Table 3.

Tissues were fixed in 4% paraformaldehyde (#50-980-495; Thermo Fisher) for 24 h and then embedded in a paraffin wax block. Sectioning with a cryostat, deparaffinization, antigen retrieval and immunohistochemistry for Ki67 (#14-5698-82; Thermo Fisher) was performed by the CRUK CI Histopathology Core using a Leica Bond III autostainer. The slides were scanned on a Leica Aperio AT2 system and subsequently analyzed in a blinded manner. H&E staining was performed by the Histology Facility at Cold Spring Harbor Laboratory.

Lung tissues were immediately fixed after harvesting, embedded in paraffin, subjected to sectioning (5 μm sections), and stained with hematoxylin and eosin (H&E) as well as with antibodies against SARS-CoV-2 spike protein, CD45+ , and CD11b, respectively. For H&E staining, lung sections were processed following standard histological routine. For indirect immunohistochemistry (IHC), lung sections were also processed following standard IHC routine slides with antigen retrieval (i.e., 15 min heat-induced with EDTA pH 6.0). Primary antibodies were used as follow: anti-spike protein at 1:200, anti-CD45+ at 1:200, and CD11b at 1:200. Taken images were scanned with Aperio AT2 system (Leica, Buffalo Grove, IL). The areas with detected spike protein, CD45+ , and CD11b, were divided by the sum of the areas corresponding to cellular structures counterstained with hematoxylin + anti-spike protein, anti-CD45+ , and anti-CD11b, respectively. Calculated ratios are represented as a % of control. All histology and IHC was performed cryptically coded sections by third party at the Inotiv facility (Boulder, CO).