Rabbit anti vegf antibody

Protein was extracted from P14 retinas from Dscam mutants and wild-type littermate controls using mem-PER Eukaryotic Protein Extraction Reagent Kit (Thermo Scientific, Rockford, IL, USA); 20 μg total protein was loaded into each lane and was separated using 10% acrylamide gel. Blots were transferred to polyvinylidene difluoride (PVDF) membranes (162-0174; Bio-Rad Laboratories, Hercules, CA, USA) and blocked in tris-buffered saline and tween 20 (TBST) (0.05% Tween) and 3% nonfat dry milk. Blots were probed with rabbit anti-VEGF antibody (1:1000; Santa Cruz Biotechnology) for 75 minutes at RT followed by four 5-minute washes in TBST at RT. Anti-VEGF was detected using goat anti-rabbit horseradish peroxidase (HRP)-linked antibody (7074, 1:25,000; Cell Signaling Technology), diluted in 3% milk. Blots were then washed four times for 5 minutes in TBST at RT. Immoblin Western Chemiluminescent HRP Substrate Kit (Millipore) was used to detect antibodies. Blots were then striped and re-probed with rabbit anti-Glyceraldehyde-3-Phosphate Dehydrogenase (anti-GAPDH) antibody (247002, 1:1000; Synaptic Systems, Goettingen, Germany) and detected with anti-rabbit HRP linked antibody (Cell Signaling Technology). Steps for anti-GAPDH were performed identical to anti-VEGF. Densitometry was performed using FIJI to calculate the relative densities between anti-VEGF and anti-GAPDH.

Human retinal endothelial cell lines (HRECs) were purchased from Ya Ji biologic technologies (Shanghai, China). Dulbecco’s modified Eagle medium (DMEM) was obtained from Thermo scientific (Hyclone, UT). Fetal Bovine Serum came from PAA laboratories (Cölbe, Germany). Trypsin-EDTA, D-glucose, and adrenomedullin_22–52 were originated from Sigma-Aldrich (St Louis, MO). The cell counting kit-8 was purchased from Dojindo (Kumamoto, Japan). Matrigel was bought from Becton Dickinson Labware (Bedford, MA). The primers were synthesized by the Shanghai biologic engineering technology service (Shanghai, China). Traces of RNA extraction kit (RNeasy Mini Kit) and reverse transcription Kit (Ominiscript RT Kit) came from Germany Qiagen Company (Qiagen, Hilden, Germany). Rabbit anti VEGF antibody was shipped from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-PI3K P110 alpha (Phosphatidylinositol 3 kinase catalyticalpha) was purchased from biolegend (San Diego, CA), and GAPDH antibody was obtained from the Beyotime Institute of Biotechnology (Shanghai, China).

After animal sacrifice, tumor tissues obtained from CT26 tumor-bearing subjects were prepared in formalin-fixed, paraffin-embedded sections. Samples were cut into 4-μm-thick serial sections, deparaffinized with xylene, and rehydrated in alcohol. Sections were subsequently submerged in antigen retrieval buffer and microwaved for antigen fixation. Sections were treated with hydrogen peroxide to block endogenous nonspecific binding activity and were incubated overnight at 4°C with diluted primary antibodies. Slides were incubated with the appropriate Horseradish Peroxidase-polymer-conjugated secondary antibodies at 37°C for 1 h. The primary antibodies that were used are as follows: mouse anti-cAMP antibody (sc73761; Santa Cruz Biotechnology Inc., Dallas, TX, USA), mouse anti-PKA antibody (sc28315; Santa Cruz Biotechnology Inc.), rabbit anti-CD31 antibody (sc-1506; Santa Cruz Biotechnology Inc.), and rabbit anti-VEGF antibody (sc-507; Santa Cruz Biotechnology Inc.). Negative control slides followed the same protocol, with the exception that primary antibodies were replaced with PBS. Subsequently, we stained sections with 3,3-diamin-obenzidine solution for 3 min and counterstained the nuclei with hematoxylin. Stained tissue slices were reviewed and independently scored by two pathologists.