Anti collagen 1 rabbit polyclonal antibody

Histological sections were prepared by standard procedures and stained with hematoxylin and eosin or immunostained for hypoxic tissue, blood vessels, or collagen-I. Pimonidazole [1-[(2-hydroxy-3-piperidinyl)-propyl]-2-nitroimidazole], injected as described earlier [47 (link)], was used as a marker of tumor hypoxia, and CD31 was used as a marker of blood vessel endothelial cells. An anti-pimonidazole rabbit polyclonal antibody (Professor James A. Raleigh, University of North Carolina, Chapel Hill, NC, USA), an anti-mouse CD31 rabbit polyclonal antibody (Abcam, Cambridge, UK), or an anti-collagen-I rabbit polyclonal antibody (Abcam) was used as primary antibody. Quantitative studies were carried out on preparations cut through the central regions of tumors, and three sections of each staining were analyzed for each tumor. Microvessels were scored as described by Weidner [28 (link)]. Fraction of pimonidazole-positive tissue and fraction of collagen-I-positive tissue were assessed by image analysis [48 (link)] and were defined as the area fractions of the non-necrotic tissue showing positive staining.

Cells were lysed with Nonidet P-40 (NP-40) lysis buffer (50 mM Tris–HCl [pH 8.0], 150 mM NaCl, 1% NP-40) containing protease inhibitor cocktail. Proteins were separated on sodium dodecyl sulfate‑polyacrylamide gel electrophoresis gels and were electroblotted onto polyvinylidene fluoride membrane (GE Healthcare Bio-sciences, Piscataway, NJ). The polyvinylidene fluoride membranes were incubated with anti-phospho-Smad2 antibody (Ser465/467), anti-phospho-extracellular-signal-regulated kinases (ERK) 1/2 monoclonal antibody (Thr202/Tyr204), anti-total-Smad2 mouse monoclonal antibody, anti-total-ERK1/2 rabbit monoclonal antibody, anti-E-cadherin rabbit monoclonal antibody, anti-vimentin rabbit monoclonal antibody, anti-N-cadherin rabbit monoclonal antibody (Cell Signaling, Danvers, MA), anti-α-SMA rabbit polyclonal antibody, anti-collagen I rabbit polyclonal antibody (Abcam), or anti-fibronectin mouse monoclonal antibody (BD Biosciences). The HRP‑conjugated goat anti-rabbit or anti-mouse IgG (GE Healthcare Bio-sciences) served as the secondary antibodies. The immunolabeled proteins were visualized by enhanced chemiluminescence. The band intensity was analyzed densitometrically by using ImageJ software (NIH).

Histological sections were prepared by standard procedures and stained with hematoxylin and eosin (HE) or immunostained for collagen I. An anti-collagen I rabbit polyclonal antibody (Abcam, Cambridge, UK) was used as primary antibody. For each of 22–28 early or late generation tumors per PDX model, quantitative studies were carried out on three sections cut through central tumor regions. The fraction of collagen I-positive tissue, defined as the area fraction of non-necrotic tissue showing positive staining for collagen I, was assessed by image analysis.