Vegfa duo set elisa kit

Secreted VEGF-A concentration was collected 24 hours and 48 hours after IR and detected in filtered conditioned media using a VEGF-A DuoSet ELISA kit according to the manufacturer's guidelines (R&D Systems; DY293B-05). The absorbance was determined with a plate reader TECAN Infinite 200 PRO at 450 nm excitation and 540 nm emission and normalized to live cell count using EVE Automatic cell counter (NanoEnTek).

The proteins were extracted using the mirVana Paris Kit and Trizol Reagent (Applied Biosystems) and quantified using the BCA Protein Assay Kit (Abcam, Cambridge, United Kingdom).
Quantification of VEGFA protein in fresh tissue samples was performed using VEGFA Duo Set ELISA Kit (R&D Systems, Minneapolis, MN, USA) following the manufacturer’s instruction. Immunohistochemistry was performed for analysis of NRF2 protein quantification. Briefly, after deparaffinization, the sections were rehydrated in a graded series of ethanol. The polyclonal rabbit anti-Nrf2 primary antibody (PA5-27882, Thermo Fisher Scientific) was used at a dilution of 1:100. After incubation, a biotinylated secondary antibody (Histostain-Plus IHC Kit, DAB, broad spectrum, 95-9943B, Invitrogen, Carlsbad, CA, USA) was used. The slides were incubated with streptavidin complex conjugated to peroxidase and 3,3′-diaminobenzidine (DAB 750118, Invitrogen) in the dark. For analysis of densitometry, the sections were photographed under a 40x objective (three fields per slide). For each sample, the cytoplasm and nucleus of epithelial cells were evaluated at 20 points equally distributed in the cytoplasm and 10 points in the nucleus.

The proteins were extracted using the RIPA Buffer (Sigma-Aldrich, San Luis, Missouri, EUA), and quantified using the Pierce ^TM BCA Protein Assay kit (Thermo Fisher Scientific, Waltham, Massachusetts, EUA). Quantification of VEGFA protein in the transfection assays with miR-612, miR-637, miR-874, and negative control was performed using the VEGFA Duo Set ELISA Kit (R&D Systems, Minneapolis, Minnesota, EUA) and Western blotting (WB) method utilizing Anti-VEGFA antibody (ab1316, Abcam, Cambridge, United Kingdom), following the manufacturer’s instruction.