Antigen retrieval citrate buffer

FTA, CTA, and DeCTA were fixed in 10% formalin, embedded in paraffin, and sliced into 5-µm sections. The sections were deparaffinized, rehydrated, washed in distilled water, and mounted on slides. The sections were subjected to histological hematoxylin and eosin (H&E) staining, 4′-6-diamidino-2-phenylindole (DAPI) staining, Masson trichrome staining, and Safranin O staining. For immunohistochemical staining, the sections were placed in antigen retrieval citrate buffer (Biogenex, Fremont, CA) in a steamer at 95°C for 10 min. Endogenous peroxidases were blocked by incubation with Peroxide Block (Innogenex, San Ramon, CA), and nonspecific binding was blocked with normal goat serum (Vector Laboratories, Burlingame, CA). The sections were incubated overnight with primary antibodies against MHC-1, MHC-II, OX62, collagen II, and collagen IV (all at 1:200; Abcam, Cambridge, MA) at 4°C. The sections were washed, and biotinylated secondary antibody was applied for 30 min, followed by treatment with streptavidin-horseradish peroxidase complex (Vectastain ABC Kit, Vector Laboratories) and diaminobenzidine solution (DAB Kit, Vector) and counterstaining with hematoxylin. The sections were dehydrated, mounted, and imaged using an IX71 microscope (Olympus, Center Valley, PA).