VEGF Antibody (C-1): sc-7269

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	-Mouse (1:2000) -Use clean forceps to gently handle the blot from the corner without creasing the membrane. Do not write on the blot with pen or marker, as the ink can fluoresce and cause background.

Protocol tips
-Mouse (1:2000) -Use clean forceps to gently handle the blot from the corner without creasing the membrane. Do not write on the blot with pen or marker, as the ink can fluoresce and cause background.

Publication protocol

The rats were sacrificed by exsanguination and the corneas harvested from the treated eyes were dissected and frozen at −70°C, then homogenized in ice-cold RIPA lysis buffer solution (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA). Following centrifugation for 5 min at 12,000 × g, the supernatants were collected and the protein concentrations were determined using the Bradford reagent (Sigma-Aldrich Chemie GmbH, Steinheim, Germany). Equal quantities of protein (15 µg/lane) from the cell lysates were separated using 8–15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto nitrocellulose membranes (Santa Cruz Biotechnology, Inc.). Membranes were incubated for 2 h in phosphate-buffered saline plus 0.1% Tween-20 and 5% non-fat skim milk to block non-specific binding. The membranes were then incubated overnight at 4°C with the following antibodies: Goat monoclonal anti-mouse VEGF (1:2,000; sc-7269; Santa Cruz Biotechnology, Inc.), goat monoclonal anti-mouse VEGF receptor (VEGFR)-1 (1:3,000; 4762; Sigma-Aldrich), goat monoclonal anti-mouse VEGFR-2 (1:1,000; 3817; Cell Signaling Technology, Danvers, MA, USA.), goat monoclonal anti-mouse matrix metalloproteinase (MMP)-9 (1:2,000; sc-21733; Santa Cruz Biotechnology, Inc.), and goat monoclonal anti-mouse β-actin (1:10,000; sc-47778; Santa Cruz Biotechnology, Inc.), which was used as a loading control. Subsequently, the membranes were incubated with the anti-mouse horseradish peroxidase-conjugated immunoglobulin G (1:10,000; sc-2005; Santa Cruz Biotechnology, Inc.), and protein bands were visualized using a SuperSignal chemiluminescence detection kit (Thermo Fisher Scientific, Inc., Rockford, Illinois, USA).

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Manufacturer protocol

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