The brains were cut into 25-μm-thick sections for immunostaining analysis. Based on our established protocol [28 (link)], the slices were blocked with PBS containing 5% BSA and incubated with 0.25% Triton-X 100. Next, the slices were incubated with primary antibodies, including rabbit anti-Factor VIII (1:200, Bioss) and rabbit anti-MMP-9 (1:200, Abcam), overnight at 4 °C, followed by incubation with the appropriate Alexa 488 or 594-conjugated secondary antibody (1:500, Jackson). The brain sections were washed with PBS and then incubated in DAPI. The brain sections were sealed with 50% glycerin and observed under a fluorescence microscope.
Alexa 488 or 594 conjugated secondary antibody
Manufactured by Jackson ImmunoResearch
Alexa Fluor 488 or 594-conjugated secondary antibodies are fluorescent-labeled antibodies that can be used to detect and visualize target proteins in various immunoassays, such as Western blotting, immunohistochemistry, and flow cytometry. These secondary antibodies are specifically designed to bind to the primary antibody, allowing for the detection and localization of the target protein of interest.
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2 protocols using alexa 488 or 594 conjugated secondary antibody
1
Immunostaining Analysis of Brain Tissue
2
Immunofluorescence Imaging of Rat Brain
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Rat brains were cut into 25-µm para n sections for immuno uorescence. The slices were treated with 0.25% Triton-X 100 for 30 min and sealed with PBS containing 5% bovine serum albumin (BSA) for 1 h. Then, the slices were directly incubated with the primary antibodies at 4°C overnight, such as rabbit anti-P2RX7 (1:200, Proteintech) and mouse anti-Claudin-5 (1:100, Invitrogen). After washing with PBS for 3 times, it was sealed with 5% BSA containing the Alexa 488 or 594-conjugated secondary antibody (1:500, Jackson). The tissue sections were washed twice in PBS and then immersed in 4′, 6-diamidino-2phenylindole (DAPI) with 20min. The sections were detected with a uorescence microscope (Eclipse 90i; Nikon, Tokyo, Japan).
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