Anti fibronectin antibody

Immunohistochemical localization of α-SMA and fibronectin was performed in endometriosis lesions as already described [68 (link),69 (link)]. The sections were incubated overnight with primary antibodies: anti-α-SMA antibody (Santa Cruz Biotechnology, CGA7) or anti-fibronectin antibody (Santa Cruz Biotechnology, sc-271098). All sections were washed with PBS and then treated as previously reported [70 (link),71 (link)]. The stained sections were observed using a Leica DM6 microscope following a typical procedure [72 (link)]. The histogram profile is related to the positive pixel intensity value obtained [73 (link)].

Glomerulosclerotic scores were evaluated by a semiquantitative method in 20 glomeruli per animal using 2-μm kidney sections stained with periodic acid-Schiff (PAS) stain^{61 (link)}. Assessment of tubulointerstitial injury was evaluated in the cortical regions using PAS staining and a semiquantitative scoring system evaluating interstitial fibrosis, inflammation, tubular atrophy, tubular dilation, debris accumulation, and cast formation in 20 tubulointerstitial areas per animal. A score of (0) for normal tubulointerstitium, (1) for injury in less than 25%, (2) for injury up to 50%, and (3) for injury in more than 50% of the biopsy specimen, as described in the previous study^{33 (link)}. Immunohistochemistry was performed with a rabbit polyclonal anti-HIF-1α antibody (1:200) (Novus Biologicals) and mouse monoclonal anti-fibronectin antibody (1:500) (Santa Cruz Biotechnology, Santa Cruz, CA, USA) as previously described^{33 (link)}. The picrosirius red stain was performed by Picrosirius Red Stain Kit (Polysciences, Warrington, PA, USA) and evaluated by optical microscope and polarizing microscope. The picrosirius red-positive area was measured using Image J by identifying the percentage of interstitial collagen positive region at x 32 magnification in five randomly selected regions^{62 (link)}. Morphometry was conducted in a blinded manner by two experienced nephrologists.

Glioma tumor tissue and adjacent normal tissue were washed with ice-cold PBS, homogenized on ice, and lysed by protein lysate (Pierce). After centrifugation, the protein concentration was measured by BCA protein assay kit (Pierce). Fifty micrograms of lysate was used for western blot. Briefly, whole cell protein extracts were separated on 10% SDS-PAGE and electroblotted onto a nitrocellulose membrane (Amersham). The blocked membrane was incubated first with anti-DKC1 polyclonal mouse antibody (Santa Cruz) or anti-beta-actin antibody (Sigma) and then with rabbit anti-mouse antibody conjugated with horseradish peroxidase (Sigma). The signal was detected using BM Blue POD Substrate (Roche). The following antibodies were used: anti-CTNNBIP1 antibody (1:200; Santa Cruz), anti-β-Catenin (t-β-Catenin) antibody (1:200; Product code: ab47426; Abcam), anti-β-Catenin (phospho T41 + S45, p-β-Catenin) antibody (1:200; Product code: ab81305; Abcam), anti-α-SMA antibody (1:200; Sigma, St. Louis, MO), and anti-fibronectin antibody (1:200; Santa Cruz). Anti-β-actin antibody (1:1000; Sigma) was used as a loading control.