Multimodal Immunofluorescence Imaging of Rat Brain

The method of double and triple immunofluorescence staining was performed as previously described [28 (link), 29 (link)]. Rats were transcardially perfused with cold phosphate-buffered solution (PBS) followed by 10% paraformaldehyde after rats were deeply anesthetized at 24 h after MCAO. The whole brains were fixed in 10% paraformaldehyde for 24 h then in 30% sucrose solution for 72 h. Coronal frozen slices (10 μm) were obtained with a cryostat (CM3050S; Leica Microsystems, Wetzlar, Germany) and permeabilized with 0.3% Triton X-100 in PBS for 30 min. Sections were blocked with 5% donkey serum for 1 h and incubated at 4 °C overnight with primary antibodies: anti-TGR5 (1:100 Abcam), anti-BRCA1 (1:100 Santa Cruz Biotechnology), anti- vWF (1:100 Abcam) and anti- CD31 (1:100 Abcam). The slices were viewed with fluorescence microscope (DMi8; Leica Microsystems, Germany) or confocal LSM 710 microscope and fluorescence intensity was quantified using ImageJ.