Mouse anti vegf sc 7269

Paraffin-embedded rat brain tissue sections were deparaffinized through xylene and ethanol series, followed by antigen retrieval as above. Immunofluorescence double staining was carried out in 1% horse serum using rabbit anti-GT198, or rabbit anti-von Willebrand factor (vWF) (A0082, DAKO, Carpinteria, CA), together with mouse anti-VEGF (sc-7269, Santa Cruz Biotechnology, Dallas, TX), or mouse CD31 (550274, BD Biosciences, San Jose, CA) or mouse α-SMA (A2547, Sigma, St. Louis, MO). CD133 was detected using rabbit anti-CD133 (ab19898, Abcam, Cambridge, MA) together with mouse anti-GT198 (1:200, Novus Biologicals, Littleton, CO). Secondary antibodies were anti-mouse or anti-rabbit Alexa Fluor-conjugated antibodies (Invitrogen, Carlsbad, CA). Sections were counterstained with DAPI. In GFP transfection assays, GFP fusions of wild type GT198 (1-217) or mutant (126-217) were transfected into U-251 cells in chamber slides. GT198 activation was induced by cotransfection of 100 nM of GT198 siRNA 5’-GUGAGUGAUGCUGACCUUCA-3’. The siRNA induces GT198 splice variants by targeting exon 4 as previously described [39 (link), 40 (link)]. GFP-GT198 in the cytoplasm was detected by fluorescence and countered stained with DAPI.

The ocular tissues including the retinal pigment epithelium (RPE) and choroid mix were isolated from enucleated eyes using a dissecting microscope to perform western blotting [18 (link)]. The primary antibodies used in the study, including rabbit anti-HGF (ab83760, Abcam, Cambridge, MA, USA), rabbit anti-p-MET (Tyr1234/1235) (3129; Cell Signaling Technology, Beverly, MA, USA), mouse anti-MET (sc-8057; Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse anti-VEGF (sc-7269, Santa Cruz Biotechnology), rabbit anti-p-VEGFR2 (Y951) (4991; Cell Signaling Technology), mouse anti-VEGFR2 (sc-393163; Santa Cruz Biotechnology), and mouse anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (sc-365062; Santa Cruz Biotechnology), were incubated with the blots overnight at 4°C. After incubation with the IRDye 680RD and IRDye 800CW secondary IgG (1 : 10,000; Sigma-Aldrich) for 2 h at room temperature, the blots were developed and quantified with the Odyssey Infrared Imaging System (Li-Cor Biosciences, Lincoln, NE, USA).