Anti tgr5

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.

The expression of proteins of interest in BMDCs was analyzed using western blotting as previously described 1 (link). The following primary antibodies were used: anti-TGR5 (Abcam, Cambridge, MA, USA), FXR (Abcam), Bax (Abcam), BCL-2 (Abcam), caspase3 (Abcam), cleaved-caspase3 (Abcam), LC3 (Abcam), Belin1(Abcam), P62 (Abcam), P65 (Abcam), Ikbα (Abcam), ERK1/2 (Santa Cruz), P38 (Abcam), JNK (Abcam), phospho-P65 (CST), phospho-Ikbα (Abcam), phospho-ERK1/2 (Santa Cruz), phospho-P38 (Abcam), phospho-JNK (Abcam), and β-actin (Abcam).

MKN45 and MKN74 cells were lysed and proteins (50 μg) were separated by 10% SDS- polyacrylamide gels, followed by electro-transfer onto a nitrocellulose membrane. For EGFR and Erk1/2 western blotting only MKN45 cells were lysed and proteins (50 μg) were separated by 8% and 12% SDS-polyacrylamide gels respectively, followed by electro-transfer onto a nitrocellulose membrane. The membranes were sequentially incubated with blocking buffer (TBS-Tween containing 5% nonfat dry milk or 5% BSA respectively) for 1 hour at room temperature, and then overnight at 4°C with one of the following antibodies: anti-TGR5 (1:2000, #ab72608 Abcam), anti-FXR (1:2000 #sc-13063 Santa Cruz Biotechnology), anti-EGFR (1:1000, #2232 Cell Signaling), and anti-phospho-EGFR antibody (1:1000, #4407 Cell Signaling), anti-Erk1/2 (P44/42 MAPK) (1:1000, #46955 Cell Signaling), anti-phospho-Erk1/2 (p-P44/42 MAPK) (1:1000, #91015 Cell Signaling). Primary antibody were detected with the horseradish peroxidase (HRP)-labeled secondary antibodies (1:10000, BioRad) for 1 hour at room temperature. After washing with TBST, protein bands were visualized by Lite Ablot TURBO (Euroclone) according to the manufacturer's instructions.