Mouse anti vegf

The liver tissues and cells were lysed in lysis buffer (Sigma-Aldrich) supplemented with Phosphatase Inhibitor Cocktail II (A. G Scientific Inc., San Diego, CA, USA) and Complete Mini Protease Inhibitor Cocktail (Roche, Basel, Switzerland). Total proteins were loaded onto SDS-PAGE gels and transferred to PVDF membranes (BIO-RAD, Hercules, CA, USA), and then incubated overnight at 4 °C with one of the following primary antibodies as indicated: rabbit anti-Col Ⅰ) (1:1000; Novus, Centennial, CO, USA), mouse anti-α-SMA (1:1000; Dako), mouse anti-VEGF (1:500; Novus), rabbit anti-ALB (1:1000; Novus), mouse anti-Cyclin D1 (1:1000; Abfrontier, Seoul, Korea), rabbit anti-hepatic nuclear factor α (HNF1α; 1:1000; Abcam Inc., Cambridge, MA, USA), mouse anti-IL-6 (1:1000; Abcam), rabbit anti-glycoprotein 130 (gp130; 1:250; Santa Cruz Biotechnology), rabbit anti-phosphorylated Stat3 (1:500; Cell Signaling), mouse anti-Stat3 (1:500; Cell Signaling), and rabbit anti-GAPDH (1:3000; Abfrontier). The membranes were washed and then reacted with a secondary antibody (horseradish peroxidase-conjugated anti-mouse IgG (1:5000; Cell Signaling) or anti-rabbit IgG (1:10,000; Cell Signaling) for 1 h at RT. The membranes were incubated using enhanced chemiluminescence reagents (Thermo Fisher Scientific., Waltham, MA, USA).

Mice treated with either IL-8 or hUCBCs 7 days after HI were dissected 14 days after injury. Isoflurane anaesthetized mice were intracardially perfused with 30 ml of saline followed by 30 ml of 4% paraformaldehyde. Their dissected brains were fixed overnight and cryoprotected in 30% sucrose. Coronal sections (10 µm) were serially collected throughout the brain and mounted on glass slides: every slide was assessed for histology and IHC.
The tissues were then washed in PBS and incubated for 1 h in blocking solution (2% normal goat serum, 0.5% Triton, PBS). The tissues were subsequently incubated for 24 h with the following primary antibodies: rabbit anti-CD31 (1:50, Abcam) and mouse anti-VEGF (1:100, Novus Biologicals). Thereafter, fluorescence-conjugated secondary antibodies (1:1,000, Alexa 488-conjugated goat anti-rabbit, Alexa Fluor 594 goat anti-mouse) were incubated with the tissue sections for 1 h at room temperature. After the sections had been washed, they were mounted in ProLong Gold reagent with DAPI (Molecular Probes, Invitrogen). To estimate vessel density, the number of vessels formed was measured using the ImageJ program after an image was obtained by fluorescence microscopy at 200× magnification.