Mouse vegf duoset elisa

Mouse VEGF DuoSet ELISA (R&D Systems, cat.no. DY493), mouse FGF basic DuoSet ELISA (R&D Systems, cat.no. DY3139), and human/mouse Total HIF‐1 alpha IC ELISA (R&D Systems, cat.no. DYC1935) were used in accordance with the 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 measured 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). The standard curve was generated using GraphPad Prism 8 (Graphpad, San Diego) with a four parameter logistic (4‐PL) curve fit. Results were interpolated and normalized to protein concentration.

The analysis of cytokine expression in tumors of the treatment groups at the protein level was done using different ELISA kits. Mouse Th1/Th2 Uncoated ELISA (Invitrogen) was used to determine the protein levels of IFN-γ, IL-2 and IL-4. IL-1α, IL-12, TGF-β, PDGF-BB, VEGF and COX-2 were measured with the Mouse IL-1 alpha/IL-1F1 DuoSet ELISA, Mouse IL-12 p70 DuoSet ELISA, Mouse TGF-β1 DuoSet ELISA, Mouse/Rat PDGF-BB DuoSet ELISA, Mouse VEGF DuoSet ELISA and Human/Mouse Total COX-2 DuoSet IC ELISA, respectively (all from R&D Systems). TARC was determined with the Mouse CCL17/TARC Quantikine ELISA Kit (R&D Systems). For protein extraction, various sections (of 50 μm thickness) from each tumor were collected and protein lysates were prepared using RIPA buffer (20 mM Tris–HCl (pH 7.2), 150 mM NaCl, 2% (w/v) NP-40, 0.1% (w/v) SDS, 0.5% (w/v) sodium deoxycholate) and cOmplete™ Protease Inhibitor Cocktail (Sigma-Aldrich) [28] (link). The total protein concentration of each tumor sample was determined using the DC protein assay (Bio-Rad Laboratories GmbH). Equal amounts of total protein (50 μg) were diluted with ELISA-Buffer (ELISA/ELISPOT Diluent, Invitrogen), or Reagent Diluent Concentrate 2 (R&D Systems), respectively. Cytokine amounts were determined according to the manufacturers' instructions.