Fitc conjugated anti rabbit igg antibody

Specific pathogen-free mice were purchased from The Jackson Laboratory (Bar Harbor, ME, USA). Animal care and experimental procedures were approved by the Animal Care Committee of Yeungnam University College of Medicine and handled according to the NIH guideline. The oxygen-induced retinopathy (OIR) mouse model was generated based on a previous report^{27 (link)}. Briefly, the newborn mice at P7 and their nursing mothers were exposed to 75% oxygen in a hyperoxic chamber (O₂ Control InVivo Cabinet, Coy Laboratory, MI, USA) for 5 days, and then were returned to room air. The anesthetized mice were injected intravitreally with 1ul of 10 mM BVT-948 or DMSO control at P12 using a micro-injector (Nanoliter 2000; World Precision Instruments, Sarasota, FL, USA) fitted with glass capillary pipettes. After 5 days, immunohistochemistry of the whole-mounted retinas was performed as previously described^{45 (link)}. Briefly, the retinal cups were incubated along with hamster anti-CD31 monoclonal antibody (2H8; Millipore Corp.) and rabbit anti-NG2 polyclonal antibody (Millipore, AB5320). After several washes, the samples were incubated for 4 h at room temperature with Cy3-conjugated anti-hamster IgG antibody (Jackson ImmunoResearch Laboratories) and FITC-conjugated anti-rabbit IgG antibody (Jackson ImmunoResearch Laboratories).

To detect expression of membrane-bound GLUTs, cells were fixed with 80% ethanol and incubated with anti-GLUT3 antibody (Abcam) and stained with the appropriate FITC-conjugated anti-rabbit IgG antibody (Jackson Immuno Research). Quantification of FITC-fluorescent intensity was performed using a FACSCanto II (BD Biosciences). Procedures for 3-OMeG uptake assay were previously described (26 (link)). LAD cells were treated with indicated TKIs for 6 h before glucose transport assay. Uptake was performed from 0.5 min to 10 min and radioactivity in the cells was quantified with Tri-Carb 3110TR low activity liquid scintillation analyzer (PerkinElmer).

To quantitatively detect the expression of membrane-bound GLUT1, cells were fixed with 80% ethanol, incubated with anti-GLUT1 antibody (Abcam), and then stained with the appropriate FITC-conjugated anti-rabbit IgG antibody (Jackson ImmunoResearch Laboratories). Quantification of FITC fluorescence intensity was performed using a FACSCanto II (BD Biosciences).