Anti dectin 1

Total proteins from the ischemic cortical area brain tissues or BV2 microglial cells were collected in equal amounts of cell lysate, and the separated proteins in the supernatant were subsequently transferred. For immunoblotting, the following primary antibodies were used: Anti-Dectin-1 (1:1,000; Abcam), anti-Syk (1:1,000; Cell Signaling Technology, Inc.), anti-p-Syk (1:1,000; Cell Signaling Technology, Inc.), anti-TNF-α (1:1,000; Cell Signaling Technology, Inc.), and anti-iNOS (1:1,000; Cell Signaling Technology, Inc.). Western blotting was performed at 6 h and 3, 5, and 7 days after ischemic stroke for the mouse tissues and at 0, 3, 6, and 12 h after OGD/R treatment and 3, 6, 12, and 24 h after LPS treatment for the BV2 microglial cells. Then, the optimal time points of 3 days for the in vivo experiments and 3 h for OGD/R treatment and 24 h for LPS treatment in vitro were selected for the subsequent experiments.

Equal amounts of protein from tissue homogenates were electrophoresed through SDS-PAGE 8–18% gradient gels. The material in the gels was blotted onto a polyvinylidene difluoride (PVDF) membrane (Bio-Rad), and the membrane was blocked with 3% BSA in TBS buffer pH 7.5 for 2 hr. After washes (TBS pH 7.5, 0.02% Tween 20), immunolabeling was performed using the primary antibody at 4°C overnight. The following primary antibodies were used: anti-dectin-1 at 1∶1000, anti-TLR2 at 1∶1500, and anti-TLR4 at 1∶500 (Abcam). Then membranes were incubated with the appropriate secondary antibodies (Cell Signaling, 1∶2000) for 2 h at room temperature. Anti-GAPDH (1∶1000 Santa Cruz Biotechnology) was used as loading control. After washing, protein bands were visualized with Chemiluminescent HRP Substrate (Millipore) for 5 min at room temperature and exposed to X-ray film (Fuji Hyperfilm). Relative changes in protein expression were estimated from the mean pixel density using Quantity One software 4.6.2 (Bio-Rad), normalized to GAPHD and presented as relative density units.