Rat anti 1 a 1 e

The following antibodies used in tissue optical clearing experiments were diluted in 0.5% bovine serum albumin (BSA), 10% dimethyl sulfoxide (DMSO), and 0.5% Triton-X-100 in PBS: rabbit anti-SARS-CoV N (Rockland Immunochemicals, #200–401-A50, 1:500), mouse anti-SARS-CoV NP (Sino Biological, #40143-MM05, 1:400), rat anti-I-A/I-E (Biolegend, #107601, 1:400), mouse anti-CD68 (Invitrogen, #MA5-13324, 1:400), rat anti-CD68 (BioLegend, #137001, 1:400), rat anti-Ki-67 (Biolegend, #652401, 1:200), rabbit anti-vWF (Dako, #A0082, 1:1,000), rabbit anti-Cleaved Caspase-3 (Cell Signaling, #9661, 1:200), and rabbit anti-Uteroglobin (Abcam, #ab40873, 1:500). Isotype control rabbit (Biolegend, #910801), rat (Biolegend, # 400602), and mouse (Biolegend, #401402) antibodies were used. Secondary antibodies were diluted in 2% donkey serum, 10% DMSO, and 0.5% Triton-X-100 in PBS at 1:1,000. All the following secondary antibodies were purchased from Invitrogen unless stated otherwise. Donkey anti-rabbit Alexa Fluor 568 (#A10042), donkey anti-mouse Alexa Fluor 568 (#A10037), donkey anti-rabbit Alexa Fluor 647 (#A31573), donkey anti-rat Alexa Fluor 647 (Jackson ImmunoResearch, #712–605-153), donkey anti-goat Alexa Fluor 647 (#A21447), donkey anti-rabbit Alexa Fluor 790 (#A11374), donkey anti-rat Alexa Fluor 790 (Jackson ImmunoResearch, #712–655-153).

Inflammatory skin infiltrates and markers were evaluated on frozen skin sections as previously described (4 (link)). Acetone-fixed frozen skin sections were blocked in Dulbecco’s PBS + 5% goat serum (catalog S-1000-20, Vector Laboratories) for 1 hour at room temperature. The fixed skin sections were incubated with rat anti-CD8 (catalog 100702, 1:100, BioLegend) and rat anti–I-A/I-E (catalog 107602, 1:100, BioLegend) overnight at 4°C, followed by incubation with Alexa Fluor 594–labeled secondary antibody (catalog A-11007, 1:500, Thermo Fisher Scientific). The endogenous biotin was blocked using a streptavidin/biotin blocking kit (catalog SP-2002, Vector Laboratories). Antifade Mountant with DAPI (catalog H-1200-10, Vector Laboratories) was used as the mounting medium. Immunofluorescence images were captured on a Zeiss LSM 700 laser scanning confocal microscope.

Small pieces of the ileum were fixed in formalin for 24 h and embedded in paraffin. Paraffin blocks were sectioned to 7 μm thickness, and slides were deparaffinized with xylenes, ethanol (100%, 95%, and 70%), and water. Antigens were retrieved in a citrate buffer at 95°C for 20 min. Slides were blocked with 1% bovine serum albumin (BSA) followed by overnight incubation with rabbit anti-EpCAM (CD326) (Invitrogen, cat#MA5-35283) and rat anti-I-A/I-E (Biolegend, cat#107601) antibodies at 4°C to stain epithelial cells and MHC class II molecules, respectively. After incubation, slides were washed and were incubated with goat anti-rabbit (Invitrogen, cat#A-11008) and goat anti-rat (Invitrogen, cat#A-11081) secondary antibodies for 1 h at room temperature. Slides were counterstained with DAPI and visualized using a Zeiss fluorescence microscope.
MHC class II molecules were quantified using the Analyze Particles function (value of parameters of size and circularity were 0.0001–0.01 and 0.50–1.00, respectively) in ImageJ software.^{57 (link)} 6–10 images were used per mouse with 3–4 mice per group.