Alexa fluor 488 conjugated goat anti rat igg

The brains were perfused with 4% paraformaldehyde in PBS (pH 7.4) and frozen. Immunohistochemistry was performed on 30 µm sections using rabbit polyclonal anti-mouse Ngb (Sigma 1:100), mouse monoclonal anti-NeuN (Chemicon; 1:200), rat monoclonal anti-PDGFRβ (Abcam; 1:100), mouse monoclonal anti-αSMA (Abcam; 1:100), mouse monoclonal anti-glial fibrillary acidic protein (Sigma; 1:200), as primary antibodies, and Alexa Fluor 488-conjugated goat anti-rat IgG (Abcam; 1:500), Alexa Fluor 594-conjugated goat anti-mouse IgG (Invitrogen; 1:500), and Alexa Fluor 546-conjugated goat anti-rabbit IgG (Invitrogen; 1:500) as secondary antibodies. Controls included omitting primary or secondary antibodies. Fluorescence signals were detected using a Zeiss LSM 800 confocal laser scanning microscope at excitation/emission wavelengths of 495/519 (Alexa Fluor 488), 556/573 (Alexa Fluor 546), 590/617 (Alexa Fluor 594), and 358/461 (DAPI) nm. In order to quantify Ngb expressed around blood vessels, we measured Ngb signals within 2 mm of all blood vessels using 512×512-pixel figures by image J.

BBB disruption could be also assessed through a one-step immunohistochemical detection of IgG²⁴ (link). The rats were transcardially perfused with PBS (pH 7.2 to 7.4, Beyotime) followed by ice-cold 4% paraformaldehyde (Servicebio, Wuhan, China). Then, rat brains were removed, post-fixed in 4% paraformaldehyde (Servicebio), cryoprotected in 15% and 30% sucrose in PBS (pH 7.2 to 7.4) (Beyotime) in turn for 2 days, and then sliced into 20 mm frozen sections on a freezing microtome (Leica, Japan). The sections were rinsed thrice with PBS (pH 7.2 to 7.4, Beyotime), followed by blocked with 10% normal goat serum (Vector Laboratories, Burlingame, CA, USA) for 1 h, and then the sections were incubated 1 h at room temperature with Alexa Fluor 488® conjugated goat anti-rat IgG (Abcam). After three rinses with PBS (pH 7.2 to 7.4, Beyotime) for 5 min each time, the fluorescent images were visualized with a VS200 Research Slide Scanner (Olympus, Tokyo, Japan), and the fluorescence intensity of lgG and area of lgG extravasation were measured by Image J software.