Horseradish peroxidase conjugated anti mouse antibody

Samples were cut into 6 μm sections after being fixed in paraformaldehyde and embedded in paraffin. Hematoxylin and eosin (H&E), safranin-O, Masson's trichrome, and Von Kossa staining were performed according to standard procedures. Two-step indirect immunohistochemical staining was performed to investigate the expression of type I, type II, and type X collagen in matrices using a primary anti-collagen type I antibody (mouse anti-rabbit, 1 : 50; Abcam, Cambridge, MA, USA), anti-collagen type II antibody (mouse anti-rabbit, 1 : 50; Acris, Herford, Germany), and anti-collagen type X antibody (mouse anti-rabbit, 1 : 50; Abcam, Cambridge, MA, USA), followed by a horseradish peroxidase-conjugated anti-mouse antibody (1 : 200 in PBS; Santa Cruz, Dallas, Texas, USA) and color development with diaminobenzidine tetrahydrochloride (Santa Cruz, Texas, USA). The sections were counterstained with hematoxylin solution (Mayer's).

Western blotting was carried out as described previously [22] (link). Lysed samples were boiled for 5 min with gel-loading buffer (0.125 M Tris-HCl, pH 6.8, 4% SDS, 10% 2-mercaptoethanol and 0.2% bromophenol blue) at a volume ratio of 1∶1. Total protein-equivalents were separated by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) using 10% acrylamide gels, and transferred to PVDF membranes at 15 V for 1 h in a semi-dry transfer system. Membranes were immediately placed in blocking buffer (5% non-fat milk) in 10 mM Tris (pH 7.5), 100 mM NaCl, and 0.1% Tween 20. Blots was allowed to block at room temperature for 1 h. Membranes were incubated with appropriate specific primary antibodies at 4°C overnight, and then treated with horse radish peroxidase-conjugated anti-mouse antibody (Santa Cruz, 1∶10,000), anti-rabbit antibody (Santa Cruz, 1∶10,000), or anti-goat antibody (Santa Cruz, 1∶10,000) at 25°C for 1 h. Antibody labeling was detected by chemiluminescence (Alpha Innotech Corporation, San Leandro, CA, USA). Pre-stained protein markers were used for molecular weight determinations.

Samples were fixed in 4% paraformaldehyde and embedded in paraffin and then cut into 8 um sections. Sections were stained with hematoxylin and eosin (H & E), safranin-O, Masson trichrome or Von Kossa. To investigate the expression of type I, type II and type X collagen in matrices and vascular endothelial growth factor (VEGF), some sections were processed for two-step indirect immunohistochemical staining as studied previously [17 (link)]. Briefly, expression of type I collagen, type II collagen, type X collagen and VEGF was detected using primary anti-collagen type I antibody (mouse anti-rabbit, 1:50; Abcam, Cambridge, MA USA), anti-collagen type II antibody (mouse anti-rabbit, 1:50; Acris, Herford Germany), anti-collagen type X antibody (mouse anti-rabbit, 1:50; Abcam) and anti-VEGF antibody (mouse anti-rabbit, 1:50; Abcam), followed by horseradish peroxidase-conjugated anti-mouse antibody (1:200 in PBS; Santa Cruz Dallas, Texas, USA) and color development with diaminobenzidine tetrahydrochloride (Santa Cruz).

Western blotting was carried out as described previously [38 (link)]. Lysed samples were boiled for 5 min in a gel-loading buffer [0.125 M Tris–HCl (pH 6.8), 4% SDS, 10% 2-mercaptoethanol, and 0.2% bromophenol blue] at a volume ratio of 1:1. Total protein equivalents were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis using 10% acrylamide gels as described by Laemmli [39 (link)], and blotted onto PVDF membranes at 100V for 1 h. Membranes were immediately placed into blocking buffer [1% nonfat milk in 10 mM Tris (pH 7.5), 100 mM NaCl, and 0.1% Tween 20] at room temperature for 30 min. Membranes were then incubated with specific primary antibodies at 25°C for 3 h, followed by horseradish peroxidase-conjugated anti-mouse antibody (Santa Cruz, 1:10,000), an anti-rabbit antibody (Santa Cruz, 1:10,000), or an anti-goat antibody (Santa Cruz, 1:10,000) at 25°C for 1 h. Antibody labeling was detected using West-zol Plus and chemiluminescence FluorchemTMSP (Alpha Innotech Corporation, San Leandro, CA, USA). Pre-stained protein markers were used for molecular weight determinations. All the raw data for the Western blotting are provided as supplementary data (S3 Data).

Western blot assays were performed as described previously with minor modification (Gershoni & Palade 1983). Cytosolic or nuclear proteins (20∼100 μg of protein) were boiled for 5 min in gel-loading buffer (60 mM Tris–HCl, pH 6.8, 2 % SDS, 25% glycerol, 10 % 2-mercaptoethanol, and 0.1 % bromophenol blue) at a volume ratio of 1:1. Samples containing the same amounts of proteins were then separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis in 6 % ∼ 15 % acrylamide gels and transferred by using a Bio-Rad western system (Bio-Rad, Hercules, CA, USA) to PVDF membranes, which were immediately placed in blocking buffer (5% non-fat milk) containing 10 mM Tris (pH 7.5), 100 mM NaCl, and 0.1% Tween 20. Membranes were then washed in TBS-Tween buffer for 30 min, incubated with specific primary antibodies (dilution 1:500 to 1:2,000, Supplementary Table 1) at 4 °C overnight, washed for 3x10-min in TBS-Tween buffer, and incubated with horseradish peroxidase-conjugated anti-mouse antibody (Santa Cruz, 1:10,000), anti-rabbit antibody (Santa Cruz, 1:10,000), or anti-goat antibody (Santa Cruz, 1:10,000) at 25 °C for 1 h. The resulting immunoblots were visualized by using Western Bright Peroxide solution (Advansta, CA, USA) and Davinch-chemi CAS-400 (Davinch-K, Seoul, Korea), according to the manufacturer’s instructions.