Discovery ultra automated stainer

Tissues were fixed in 10 % Neutral Buffered Formalin with two changes, for a minimum of 7 days according to IBC-approved SOP. Tissues were processed with a Sakura VIP-6 Tissue Tek, on a 12-hour automated schedule, using a graded series of ethanol, xylene, and PureAffin. Embedded tissues were sectioned at 5 μm and dried overnight at 42°C prior to staining with hematoxylin and eosin. Specific staining was detected using SARS-CoV/SARS-CoV-2 nucleocapsid antibody (Sino Biological cat#40143-MM05) at a 1:1000 dilution, CD4 antibody (abcam cat#ab133616) at a 1:100 dilution, CD8 antibody (Sino Biological cat#10980-T24) at a 1:500 dilution, and CD20 (Thermo Scientific cat#RB-9013) at a 1:250 dilution. The tissues were processed for immunohistochemistry using the Discovery Ultra automated stainer (Ventana Medical Systems) with a ChromoMap DAB kit (Roche Tissue Diagnostics cat#760–159).

Necropsies and tissue sampling were performed according to IBC-approved protocols. Tissues were fixed for a minimum of 7 days in 10% neutral buffered formalin with 2 changes. Tissues were placed in cassettes and processed with a Sakura VIP-6 Tissue Tek, on a 12-hour automated schedule, using a graded series of ethanol, xylene, and ParaPlast Extra. Prior to staining, embedded tissues were sectioned at 5 μm and dried overnight at 42°C. Using GenScript U864YFA140–4/CB2093 NP-1 (1:1000) specific anti-CoV immunoreactivity, CD3 (Predilute) (Roche Tissue Diagnostics #790–4341), and PAX5 (1:500) (Novus Biologicals #NBP2–38790) were detected using the Vector Laboratories ImPress VR anti-rabbit IgG polymer (# MP-6401) as the secondary antibody. Iba-1 (1:500) (abcam #ab5076) was detected using Roche Tissue Diagnostics OmniMap anti-goat multimer (#760–4647) as the secondary antibody. The tissues were stained using the Discovery Ultra automated stainer (Ventana Medical Systems) with a ChromoMap DAB kit Roche Tissue Diagnostics (#760–159).

Tissues were fixed in 10% neutral buffered formalin for 48–72 h and transferred into 30% sucrose and embedded in paraffin. Embedded tissues were sectioned at 5 μm and dried overnight at 42°C before staining. Specific anti-CoV immunoreactivity was detected using an SCV2 nucleoprotein antibody (GenScript) at a 1:1,000 dilution. The secondary antibody was the ImmPRESS-VR Anti-Rabbit IgG Polymer (cat. no. MP-6401; Vector Laboratories). The tissues were then processed for immunohistochemistry using the DISCOVERY ULTRA automated stainer (Ventana Medical Systems) with a ChromoMap DAB Kit (cat. no. 760-159; Roche Tissue Diagnostics). All tissue slides were evaluated by a board-certified veterinary pathologist.

At time of necropsy, lungs were dissected and insufflated with 10% neutral buffered formalin. The skull was sectioned and lungs and skull sections submerged in 10% neutral buffered formalin for a minimum of 7 days with 2 changes. Tissues were placed in cassettes and processed with a Sakura VIP-6 Tissue Tek, on a 12-hour automated schedule, using a graded series of ethanol, xylene, and ParaPlast Extra. Prior to staining, embedded tissues were sectioned at 5 µm and dried overnight at 42 °C. Specific anti-CoV immunoreactivity was detected using Sino Biological Inc. SARS-CoV/SARS-CoV-2 N antibody (Sino Biological cat#40143-MM05) at a 1:1000 dilution. The secondary antibody was the Vector Laboratories ImPress VR anti-mouse IgG polymer (cat# MP-7422). The tissues were then processed for immunohistochemistry using the Discovery Ultra automated stainer (Ventana Medical Systems) with a ChromoMap DAB kit (Roche Tissue Diagnostics cat#760–159). Sections were scored by a certified pathologist who was blinded to study groups. Sections from mock-infected hamsters came from historical controls.

Histopathology and immunohistochemistry were performed on hamster lung tissues. Tissues were fixed in 10 % Neutral Buffered Formalin with two changes, for a minimum of 7 days according to IBC-approved SOP. Tissues were placed in cassettes and processed with a Sakura VIP-6 Tissue Tek, on a 12-hour automated schedule, using a graded series of ethanol, xylene, and PureAffin. Embedded tissues were sectioned at 5 μm and dried overnight at 42°C prior to staining. Specific anti-CoV immunoreactivity was detected using Sino Biological Inc. SARS-CoV/SARS-CoV-2 nucleocapsid antibody (Sino Biological cat#40143-MM05) at a 1:1000 dilution. The secondary antibody was the Vector Laboratories ImPress VR anti-mouse IgG polymer (cat# MP-7422). The tissues were then processed for immunohistochemistry using the Discovery Ultra automated stainer (Ventana Medical Systems) with a ChromoMap DAB kit (Roche Tissue Diagnostics cat#760–159). The tissues slides were scanned with the Aperio ScanScope XT (Aperio Technologies, Inc.) and the entire section analyzed with the ImageScope Positive Pixel Count algorithm (version 9.1)^{37 (link)}. All tissue slides were analyzed by a board-certified veterinary pathologist.