Immunohistochemical Profiling of Brain Injury

Immunohistochemistry was performed as previously described^{54 (link), 55 (link)}. Free floating, fixed brain sections with 40-μm thickness underwent fluorescent immunohistochemical staining using the following primary antibodies: anti-glial acidic fibrillary protein (GFAP) (OPA1-06100, Thermo Fisher, 1:1000 dilution), anti-CD45 (MCA1031G, BioRad, 1:500 dilution), anti-mouse fibrinogen (ab34269, Abcam, Cambridge, UK, 1:1000 dilution), and anti-claudin 5 (ab15106, Abcam, Cambridge, UK, 1:500 dilution). The secondary antibodies used included Alexa Fluor 488 Goat Anti-Rabbit IgG (A-11008), Alexa Fluor 488 Goat Anti-rat IgG (A-11006), and Alexa Fluor 350 Goat Anti-rat IgG (A-21093, Invitrogen; 1:500 dilution). Stained sections were imaged using a Zeiss LSM5 confocal scanning microscope system and analyzed using the Zeiss LSM Image software (Zeiss Ltd., Jena, Germany). Another cohort of brain sections underwent 3,3′-Diaminobenzidine (DAB) staining with anti-mouse fibrinogen (ab34269, Abcam, Cambridge, UK, 1:3000 dilution). Stained sections were imaged using a Nikon E-600ME microscope and analyzed using MetaMorph software. Immunohistochemical images were taken of the left cerebral cortex (ipsilateral to the lesion).