Anti collagen type 4 antibody

Individual PFC specimens were removed from −80°C, covered in OCT (Tissue Tek) in a cryomold, and allowed to set for at least two hours at −80°C. Tissue blocks were then cut on a cryostat into 20 μm sections and mounted onto a SuperFrost slide. Tissue sections were fixed using 4% PFA (Electron Microscopy Services) in PBS for one hour, then incubated in the blocking buffer (5% Normal Donkey Serum, 0.5% TritonX in PBS) for 24 hours at 4°C. We then used TrueBlack^® Lipofuscin Autofluorescence Quencher (Biotium) to remove background fluorescence according to the manufacturer’s protocol (Protocol 1, Pretreatment with TrueBlack). Tissue sections were incubated with primary antibodies for 3 days at 4°C and then washed with PBS three times, followed by incubation with secondary antibodies for 4 hours, and then finally were mounted with Fluomount-G. Sections were imaged using a Zeiss LSM 900 confocal microscope at 20X or 63X magnification. Images were quantified in FIJI, QuPath, and Imaris by an investigator blinded to diagnosis. For primary antibodies we used Anti-Collagen Type IV Antibody (Sigma-Aldrich #AB769, used at 1:125 concentration) and Lectin-488 (Vector Laboratories #DL-1174, used at 1:125 concentration).