Rabbit anti ccn2 ctgf

Total proteins were extracted from mouse livers or cultured cells in RIPA buffer containing proteinase inhibitors (Sigma). Total protein lysates (50 µg) were boiled in 1x Laemmli buffer containing 5% β-mercaptoethanol, separated in SDS-PAGE gel, and electro-transferred onto polyvinylidene difluoride membrane for immunoblotting. Primary antibodies included rabbit anti-Ccn2/Ctgf (Abcam), rabbit anti-Slit2 (Proteintech), rabbit anti-aSMA (Proteintech), rabbit anti-Collagen (Proteintech), rabbit anti-p-AKT (Cell Signaling Technology, Danvers, MA, USA), rabbit anti-AKT (Cell signaling), rabbit anti-p-PI3K (Cell signaling), rabbit anti-PI3K (Cell signaling), and rabbit anti-GAPDH (Abcam). Detection was carried out using horseradish peroxidase-conjugated secondary antibodies (Santa Cruz biotechnologies) and the ECL Plus kit (Amersham Biosciences, Piscataway, NJ, USA).

Human HCC sections were obtained from BioChain (Newark, CA). Mouse and rat livers were fixed in 4% paraformaldehyde. Histological analyses were performed with standard protocols using OCT or paraffin-embedded Sect. (6 μm). Primary antibodies used were rabbit anti-hepatocyte nuclear factor (Hnf)4a (Santa Cruz biotechnologies, Dallas, TX, USA), rabbit anti-Ccn2/Ctgf (Abcam, Cambridge, United Kingdom), and rabbit anti-cytokeratin (CK)19 (Abcam). Detection was carried out according to the manufacturer’s instructions using the ABC-Elite kit with ImmPACT DAB substrate (Vector Laboratories, Burlingame, CA, USA). Collagen deposition was measured by Sirius Red staining or Trichrome blue according to previous publication(Pi et al. 2015 ). Alexa Fluor® 596 conjugated a smooth muscle actin (SMA) antibody (Abcam) was also used for double staining with the Ccn2/Ctgf rabbit antibody (Abcam). For estimation of stained positive areas, images were captured with CellSens software using an Olympus BX 51 upright fluorescence microscope outfitted with an Olympus DP80 camera, Plan Fluorite objectives and a LED transmitted light source (Olympus, Waltham, MA, USA). DAB stained areas were quantified from 10 random fields of images using Image J software (http://rsb.info.nih.gov/ij/) and IHC profiler according to published methods (Varghese et al. 2014 (link)).