Rabbit anti mouse ng2 antibody

Tissue whole-mount staining was performed according to our previously described method (17 (link)). A rat anti-mouse CD31 antibody (BD Pharmingen; 1:200) and a rabbit anti-mouse NG2 antibody (Millipore; 1:200) were used as primary antibodies. The secondary antibodies included a Cy3- or Cy5-conjugated goat anti-rat IgG antibody (Invitrogen; 1:400) and a donkey anti-rabbit Cy5 antibody (Invitrogen; 1:400). Stained tissues were mounted in a Vectashield mounting medium (Vector Laboratories). Images were captured with a confocal microscope (Nikon D-Eclipse C1, Nikon Corp.). Quantitative analyses were performed from at least 10 different random fields using the Adobe Photoshop CS software program.

Murine tumour tissue samples were snap-frozen and prepared for microscopical analyses as described (Coutelle et al, 2015 (link)). As primary antibody for staining of endothelial cells, pericytes and type IV collagen, rat monoclonal anti-mouse CD31 antibody (PEACAM-1; 1 : 50; clone MEC 13.3, BD Pharmingen, Heidelberg, Germany), Cy3-conjugated mouse monoclonal anti-α-smooth muscle actin antibody (1 : 100; α-smooth-muscle-actin (α-SMA) clone 1 A4, Sigma-Aldrich, Munich, Germany), rabbit anti-mouse NG2 antibody (1 : 200; Millipore, Darmstadt, Germany) and rabbit polyclonal anti-type IV collagen antibody (1 : 100; Clone ab6586, Abcam, Cambridge, UK) were used, respectively. The CD31 antibody was detected by Alexa Fluor 488-conjugated polyclonal goat anti-rat antibody and the NG2 and collagen IV antibody were detected by Alexa Fluor 594-conjugated goat anti-rabbit antibody (1 : 500; Molecular Probes, Eugene, OR, USA). Immunohistochemical staining of human paraffin-embedded tumour sections was performed using the BOND MAX from Leica (Wetzlar, Germany) according to the protocol of the manufacturers. As a primary antibody Collagen IV clone CIV 22 mouse monoclonal, Dako (M0785; Glostrup, Denmark) at a dilution of 1 : 50 was used.