Rabbit anti shh

The brain tissues from the BALB/c mice were snap-frozen by immersion in isopentane chilled to −70°C, and the tissues were then immediately mounted in OCT medium. The samples were stored at −80°C until sectioning with a Microm OMV cryostat to 10–15 μm. The frozen tissue sections were fixed in 2% (w/v) PFA (paraformaldehyde) and permeabilized in 0.5% (v/v) Triton X-100 in PBS before incubation with the purified antibodies (rabbit anti-Shh (Santa Cruz Biotechnology Inc., USA, 1 : 50) and rabbit anti-GFAP (Abcam, UK, 1 : 500)) for 2 h at room temperature or overnight at 4°C. After incubation, the sections were incubated with the appropriate secondary antibodies (DyLight 488-594-conjugate anti-rabbit IgG) (Jackson ImmunoResearch Inc., UK, 1 : 1000) for 1 h at room temperature. For nuclear counterstaining, the sections were stained with DAPI. The stained sections were then examined and photographed under a light microscope.

Brain tissues from BALB/c mice were snap-frozen by immersing in isopentane chilled to −70 °C and immediately mounted in OCT medium. The samples were then stored at −80 °C until sectioned by Microm OMV cryostat to 10–15 mm. The frozen tissue sections were fixed in 2% (w/v) paraformaldehyde (PFA) and permeabilized in 0.5% Triton X-100 in PBS before incubation with purified antibodies (Rabbit anti-Shh (1:50)) (Santa Cruz Biotechnology Inc., USA), rabbit anti-GFAP (1:500) (Abcam, UK), rabbit anti-SOD (1:1,000) (Sigma-Aldrich, USA) and rabbit anti-Catalase (1:1,000) (Sigma-Aldrich, USA) for 2 h at room temperature or overnight at 4 °C. After incubation, the sections were incubated with appropriate secondary antibodies (DyLightTM 488-594-conjugate anti-rabbit IgG) (1:1,000) (Jackson ImmunoResearch Inc, UK) for 1 h at room temperature. For nuclear counterstaining, the sections were stained with DAPI. The stained sections were then examined and photographed under a light microscope.

The levels of the apoptosis-related proteins Bax and Bcl-2, GFAP, and Shh were measured by 12.5% SDS-PAGE. A semidry transfer unit (Bio-Rad, Hercules, CA, USA) was used to transfer the proteins from the gel to a nitrocellulose membrane at 0.04 mA for 50 min. The membrane was washed with TBS/T three times and then with blocking buffer. The primary antibodies were rabbit anti-Shh (1 : 100) (Santa Cruz Biotechnology Inc., USA), rabbit anti-Bax (1 : 500) (Santa Cruz Biotechnology Inc.), rabbit anti-Bcl-2 (1 : 500) (Santa Cruz Biotechnology Inc.), rabbit anti-GFAP (1 : 1,000) (Abcam, UK), rabbit anti-GRP78 (1 : 1000) (Sigma-Aldrich), and mouse anti-β-actin (1 : 5000) (Sigma-Aldrich). After incubation with these antibodies at 4°C overnight, the membranes were then washed three times before incubation with the corresponding secondary antibodies (goat anti-rabbit IgG 1 : 10,000 and rabbit anti-mouse IgG 1 : 10,000) (Sigma-Aldrich) for 45 min. The membranes were then washed with TBS/T three times and incubated with a mixture of stable peroxide solution (500 μL) and enhanced solution (500 μL) in the dark for 5 min. The ImageJ image analysis software (http://rsb.info.nih.gov/ij/index.html) was used to quantitate and compare the concentrations of the target proteins (GFAP, Shh, Bax, Bcl-2, and GRP78) and the control (β-actin).