Ab9704

PDGF-BB and p-AKT liver content were detected using “avidin-biotin-peroxidase (DAB, Sigma Chemical Co.)” on paraffin slices of the liver of the control and all treatment groups, similar to the technique described by [35 (link)]. Tissue sections were incubated with a monoclonal antibody for PDGF-BB and p-AKT (Abcam, Cambridge, MA, USA, ab9704 and ab8805 respectively) at 1:200 and 1:100 dilutions respectively; and reagents required for the avidin-biotin-peroxidase (Vactastain ABC peroxidase kit, Vector Laboratories) method for the detection of the “antigen-antibody complex.” Each marker expression was visualized by the chromogen “3,3 -diaminobenzidine tetrahydrochloride (DAB, Sigma Chemical Co.)”. Quantification of the positive brown area of each marker’s expression was implemented as an optical density in 7 high-power microscopic fields using image analysis software (Image J, 1.46a, NIH, USA).

Mouse skin tissues were fixed with 4% paraformaldehyde for overnight. Then, the tissues were washed, sealed with normal saline of 0.01 M phosphate buffer for three times, and then sealed with 10% goat serum (C0265, Beyotime, Shanghai, China) at room temperature for 30 min. After that, transforming growth factor-β1 (TGF-β1) (1:200, ab92486, Abcam), vascular endothelial growth factor (VEGF) (1:200, ab2350, Abcam), and platelet-derived growth factor (PDGF-BB) (1:200, ab9704, Abcam) were incubated with tissues at 4 °C for overnight. The secondary antibody and 4′,6-diamidino-2-phenylindole were incubated at room temperature in the dark for 1 h, and glycerol was fixed. The confocal laser scanning microscope was used for analysis (LSM, FV1000; Olympus Corp., Tokyo, Japan).

To neutralize cytokines in HUVEC (phECs) conditioned media, antibodies against human TGFβ (Abcam, ab27969), FGF2 (Millipore, 05-117), IL8 (Abcam, ab10769), IL6 (Abcam, ab6672), IGF1 (Abcam, ab9572), VEGFa (Millipore, 07-1420), PDGFβ (Abcam, ab9704) and SDF1 (Abcam, ab10395) were used. To neutralize cytokines in pmECs conditioned media, antibodies against mouse TGFβ (Abcam, ab64715), FGF2 (Abcam, ab33103), VEGFa (R&D, AF-493-NA), IL6 (R&D, AF-406-NA), IL-1a (R&D, AB-400-NA), and IGF1 (R&D, AF791) were used. See the Supplemental Experimental Procedures for details.