Anti shh sc 9024

The cerebellums of embryos were removed by dissection and were homogenized with lysis buffer containing 20 mM Tris-HCl (pH7.5), 150 mM NaCl, 1 mM Na₂EDTA, 1 mM EGTA, 1% NP-40, 1% SDS, 2.5 mM sodium pyrophosphate, 1 mM Na₂VO₄, and Complete Protease Inhibitor Cocktail (Roche Applied Bioscience, Mannheim, Germany) per manufacturer's instructions. Protein extracts were resolved by SDS-PAGE and transferred to nitro cellulose membranes. These membranes were probed with anti-SHH (sc-9024, 1∶500; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and anti-HSP70 (#4872, 1∶1000; Cell Signaling, Danvers, MA, USA) antibodies. Each blot was visualized using a SuperSignal West Pico Chemiluminescent detection kit (Thermo Scientific, Rockford, IL, USA) per manufacturer's instructions and quantified using ImageJ software (http://rsbweb.nih.gov/ij/). The expression level of SHH was normalized by the intensity of the HSP70 band in the same blot. Data from three cerebellums of each genotype were used for quantification.

Liver specimens were fixed 10% neutral buffered formalin, embedded in paraffin, and cut into 4 μm sections. The specimens were deparaffinized, hydrated, and stained usual method with standard hematoxylin and eosin staining (H&E) to examine morphology and Sirius Red staining to assess fibrosis. For immunohistochemistry (IHC), the sections were incubated for 10 min. in 3% hydrogen peroxide to block endogenous peroxidase. Antigen retrieval was performed by heating in 10mM sodium citrate buffer (pH 6.0) for 10 min. The sections were blocked in Dako protein blocking solution (X9090; Dako Envision, Dako Corp., Carpinteria, CA, USA) for 30 min. and incubated with primary antibodies, anti-Ubiquitin (sc-8017; Santa Cruz Biotechnology, Inc., CA, USA), anti-active Caspase-3 (AF835; R&D systems, Minneaoplis, MN, USA), anti-Ki-67 (NCL-KI67-MM1; Leica Microsystems, Newcastle, UK), anti-Shh (sc-9024; Santa Cruz Biotechnology, Inc., CA, USA), anti-Gli2 (GWB-CE7858; Genway Biotech, Inc., San Diego, CA, USA), or non-immune sera to demonstrate staining specificity at 4 °C overnight. Polymer horseradish peroxidase (HRP), anti-rabbit (K4003; Dako, CA, USA), or anti-mouse (K4001; Dako, CA, USA) was used as secondary antibodies. 3,3′-Diaminobenzidine (DAB) (K3466; Dako, CA, USA) was employed for the detection procedure.

Liver tissues were homogenized and digested in 1×RIPA buffer containing a protease inhibitor and phosphatase inhibitor cocktail solution (Thermo Scientific, Waltham, MA, USA). Western blotting experiments were performed following a standard protocol. The primary antibodies used were anti-SHH (SC-9024; Santa Cruz Biotechnology, Dallas, TX, USA), anti-Gli1 (ab167388; Abcam, Cambridge, UK), anti-Gli2 (ab26056; Abcam, Cambridge, UK), and anti-GAPDH (# 2118; Cell Signaling Technology, Danvers, MA, USA).