Mouse fgf basic duoset elisa

Mouse VEGF DuoSet ELISA (R&D Systems, cat.no. DY493), mouse FGF basic DuoSet ELISA (R&D Systems, cat.no. DY3139), human/mouse Total HIF-1 alpha IC ELISA (R&D Systems, cat.no. DYC1935) were used in accordance to manufacturer’s instructions. Pulverized tissue was dissolved in Lysis Buffer #11 (50 mM Tris pH 7.4, 200 mM NaCl, 10% (w/v) Glycerol, 3 mM EDTA, 1 mM MgCl₂, 20 mM β-glycerophosphate, 25 mM NaF, 1% Triton X-100) with protease inhibitor cocktail set III (Millipore-Sigma, Burlington, MA cat.no. 539134) by sonication. Total protein concentration was measure using the Pierce BCA protein assay kit with a bovine serum albumin standard according to the manufacturer’s instructions (Thermo-Fisher Scientific, Waltham, MA cat.no. 23225). ELISA was performed in a 96-well plate format and the optical density was determined at 450nm and corrected at 540nm using a FlexStation 3 microplate reader (Molecular Devices, San Jose, CA). The standard curve was generated using GraphPad Prism 8 (Graphpad, San Diego; CA) with a four parameter logistic (4-PL) curve-fit. Results were interpolated and normalized to protein concentration.