The brain tissue was post-fixed (4% paraformaldehyde for 72 h), dehydrated, embedded in paraffin, and sectioned at 4-μm thickness. The sections were dried at 40°C, followed by deparaffinization and rehydration using a series of ethanol solutions. The hematoxylin and eosin (H&E) staining was performed according to the manufacture’s protocol.
For in vivo protein evaluation, paraffin-embedded sections were deparaffinized and rehydrated as described above. Immunofluorescence staining was performed as previously described [13 (link),23 (link)]. The primary antibodies used in the present study were against claudin-5 (1: 200; Genetex, Irvine, CA, USA), ZO-1 (1: 100; Novus Biological, Centennial, CO, USA), occludin (1: 200; Abcam, Cambridge, MA, USA), or β-catenin (1: 100, CST, Danvers, MA, USA), followed by incubation with goat anti-rabbit or goat anti-mouse secondary fluorescein isothiocyanate-labeled antibody (1: 500; ZhongShan Golden Bridge Bio-technology, Beijing, China). Cellular nuclei were counterstained with Hoechst 33258 (Beyotime-Bio, Shanghai, China). The samples were imaged under ×200 magnification with a Zeiss Imager A2 (Zeiss, Jena, Germany).
For in vivo protein evaluation, paraffin-embedded sections were deparaffinized and rehydrated as described above. Immunofluorescence staining was performed as previously described [13 (link),23 (link)]. The primary antibodies used in the present study were against claudin-5 (1: 200; Genetex, Irvine, CA, USA), ZO-1 (1: 100; Novus Biological, Centennial, CO, USA), occludin (1: 200; Abcam, Cambridge, MA, USA), or β-catenin (1: 100, CST, Danvers, MA, USA), followed by incubation with goat anti-rabbit or goat anti-mouse secondary fluorescein isothiocyanate-labeled antibody (1: 500; ZhongShan Golden Bridge Bio-technology, Beijing, China). Cellular nuclei were counterstained with Hoechst 33258 (Beyotime-Bio, Shanghai, China). The samples were imaged under ×200 magnification with a Zeiss Imager A2 (Zeiss, Jena, Germany).