IDISCO

SARS-CoV-2 infected and non-infected Syrian hamster lungs were provided in 10% formalin, for longer storage hamster lungs were kept in PBS and 0.01% sodium azide. Dehydration of the lungs was performed by washing with PBS for 1.5 hours, followed by 50% methanol for 1.5 hours, 80% methanol for 1.5 hours, and as of last 100% methanol for 1.5 hours on a tilting laboratory shaker. Bleaching was performed overnight at 4°C in 90% methanol (100% v/v) and 10% H₂O₂ (30% v/v). Tissues were rehydrated with 100% methanol for 1 hour, followed by 100% methanol for 1 hour, 80% methanol, 50% methanol, and 1x PBS for 1 hour on a tilting shaker. Syrian hamster lungs were subsequently blocked for 24 hours at room temperature (22°C) in 1x PBS with 0.2% gelatin, 0.5% triton-x-100, and 0.01% sodium azide (PBSGT) on a tilting shaker. Hamster lungs were stained with primary/secondary antibodies possessing an alexa555, alexa647, or alexa750 dye. After blocking samples were incubated for 120 hours with the primary antibody in PBSGT with 0.1% saponin (S2149, Sigma-Aldrich) on a shaking incubator at 200 rpm, following 120 hours incubation, hamster lungs were washed with PBSGT six times for 1 hour on a tilting shaker. Post-PBSGT wash, samples stained with primary antibodies were incubated with secondary antibodies that possess alexa555, alexa647 or alexa750 dyes, the antibodies were diluted in PBSGT with 0.1% saponin and filtered with 0.45 μm filter for potential antibody aggregates. Hereafter samples were incubated for 120 hours at room temperature on a shaking incubator at 200 rpm. After staining washing was performed with PBSGT six times for 1 hour on a tilting shaker. Immunostained samples were subsequently treated with 50% methanol for 24 hours, 80% methanol for 24 hours, 100% methanol for 24 hours followed by 100% methanol for 24 hours on a tilting shaker for dehydration of the lungs. Hereafter lipid solubilization was performed with dichloromethane (Thermofisher, 402152) for 40 minutes after which refractive index matching and optical clearing were performed overnight with dibenzyl ether (108014, Sigma-Aldrich).