Goat anti tlr4

According to our previous methods [14] , mice were deeply anesthetized with an overdose of iso urane and transcardially perfused with 0.01 M phosphate buffered saline (PBS, pH 7.4) for 5-10 min followed by 4% paraformaldehyde in 0.1 M PBS for 15-20 min. Whole brains were removed and post-xed 4% paraformaldehyde for 24 h followed by 30% sucrose solution with 4% paraformaldehyde at 4 °C. Coronal cryosections were collected. Primary cortical neurons were xed with 4% p-formaldehyde. The sections were incubated with the primary antibodies in 1% bovine serum albumin overnight at 4 °C: rabbit anti-β III Tubulin (1:1000, Abcam), mouse anti-NMDAR2B (1:1000, Abcam) and goat anti-TLR4 (1:500, Santa Cruz). The secondary antibodies, Cy3-, 647-or 488-conjugated (1:500, Jackson ImmunoResearch) were subsequently added (2 h, 37 °C), respectively. Sections were counterstained with 4',6-diamidino-2phenylindole (Sigma-Aldrich, St. Louis, MO, USA) and were viewed using a confocal laser-scanning microscope (Leica TCS-SP2, Heidelberg, Germany) and analyzed with Leica imaging software.

Paraffin-embedded sections (7-mm thick) were examined using the following primary antibodies: mouse anti-NF200
(1:400; Sigma), mouse antieglial fibrillary acidic protein (GFAP) (50 mg/mL; Progen, Heidelberg, Germany), goat anti-iba1 (1:400; Abcam, Cambridge, England), goat anti-TLR4 (2 mg/mL; sc-16240; Santa Cruz Biotechnology, Santa Cruz, CA), and rabbit antieNF-kB p65 (1:500; catalog number 8242; Cell Signaling, Danvers, MA). The antibodies against TLR-4 18,24,30,31 and NF-kB 32, 33 have been widely used and described elsewhere. Stained sections were examined using a microscope (BX51; Olympus Corporation, Tokyo, Japan) connected to a DP70 camera (Olympus). Images were processed and viewed using DP manager software version 2.2. 1.195 (Olympus). Quantitative analysis of the stained region was performed using ImageJ software version 1.46r (NIH, Bethesda, MD; http://imagej.nih.gov/ij).

Supernatants obtained after homogenization of cell pellets in lysis buffer of protein concentration 1.5 mg/ml were separated on the 5-20 % SDS-PAGE under reducing conditions. Separated proteins were transferred onto nitrocellulose membranes blocked with Tris-buffered saline with 0.1 % Tween 20 (TBS) (Golias et al. 2013) . Blocked membranes were incubated with a primary antibody (Ab) diluted in TBS containing 0.1 % defatted milk (Promil, Nový Bydžov, Czech Republic) for 1 h at room temperature. Primary Abs: goat anti-IL-18 diluted 1:5,000, goat anti-TLR-2 and goat anti-TLR-4, both diluted 1:500 (Santa Cruz Biotechnology, Santa Cruz, CA, USA); Control recombinant rat IL-18 protein (R&D Systems, Minneapolis, MN, USA); rabbit anti-caspase-1 (Cell Signaling Technology, Danvers, MA, USA) diluted 1:1,000. The membranes were incubated with peroxidase-conjugated secondary Ab: donkey anti-goat IgG, goat anti-rabbit IgG (Cell Signaling Technology) diluted 1:5,000 with TBS containing 1 % milk. Signal was detected using the West Femto Maximum Sensitivity Substrate kit (Pierce, Rockford, IL, USA) and exposed onto films (Santa Cruz Biotechnology).