Double-IF Analysis of BBB Markers

Double-IF was performed as previously described [29 (link)]. Briefly, 15-μm-thick, frozen brain slices were washed three times with phosphate-buffered saline (PBS) and then antigen retrieval was performed in a microwave oven for 10 min in 0.01 M citrate buffer (pH 6.0). After blocking for 60 min in goat serum, the slices were incubated with primary antibodies (CD31, BD Bioscience, #562939; ZO-1, Thermo Fisher #PA5-85256; occludin, Thermo Fisher #40-4700; claudin-5, Thermo Fisher #34-1600) overnight at 4 °C. The slices were washed three times within PBS and then the corresponding secondary antibodies (DyLight 488, Abbkine; DyLight 549, Abbkine) were provided at 37 °C for 60 min. Finally, the slices were stained with DAPI (Thermo Fisher #62248) for 4 min. After washing and mounting, the sections were analyzed with an Olympus FV3000 confocal laser scanning microscope.