Hrp conjugated streptavidin

The rabbit anti-CD146 polyclonal antibody and mouse anti-CD146 monoclonal antibody AA1 and AA4 were generated in our laboratory (Zhang et al., 2008 (link)). Rat anti-mouse CD146 (clone ME-9F1) was purchased from BD Biosciences. Anti-mouse Tek-PE and anti-mouse CD31-APC antibodies were purchased from Tianjin Sungene Biotech Co., Ltd. Antibodies specific for phospho-p38 MAPK, p38 MAPK, phospho-NF-κB p65 (Ser536), phospho-ERK, ERK, AKT, were purchased from Cell Signaling Technology. Antibodies specific for I-κB were from Beijing Zhong Shan-Golden Bridge Biological Technology CO., LTD. Antibodies against phosphor-VEGFR-2 (Tyr 1214) and phospho-AKT (Ser 473) were purchased from Signalway Antibody. Antibodies specific for CD31 and GAPDH were purchased from Abcam. HRP-conjugated goat anti-mouse or rabbit IgG were purchased from GE Healthcare. Enhanced chemiluminescence assay kits were purchased from Pierce. Biotin-conjugated secondary antibodies and HRP-conjugated streptavidin were purchased from Dianova. Goat serum and the DAB substrate system were purchased from Santa Cruz Biotechnology. DAPI was purchased from Roche. Fluorescein isothiocyanate-dextran was purchased from Sigma. Growth factor-reduced Matrigel and collagenase were purchased from BD Biosciences. VEGF-A was purchased from Upstate Biotechnology.

Atherosclerotic plaque morphology and collagen content was examined in 8-μm-thick sections stained with hematoxylin and eosin and Masson's trichrome, respectively. For 3,3′-diaminobenzidine staining, paraffin-embedded tissue sections were deparaffinized and stained first with an antibody specific for CD146 (AA4), followed by a biotin-conjugated secondary antibody (1:1 000), and then by HRP-conjugated streptavidin (Dianova, Rodeo, CA). Macrophages were identified using anti-Mac-3 (a marker for murine macrophages). The sections were counterstained with hematoxylin. The number of macrophages per unit area was measured in at least 10 random lesions from the carotid artery. For immunofluorescence, sections were deparaffinized and stained with antibodies specific for CD68, Mac-3 or CD146 followed by fluorescence-labeled secondary antibodies. The nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI). The sections were visualized using a confocal laser scanning microscope (Olympus, Tokyo, Japan).