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Extended reaction time Reagent Preparation Prepare 4 N NaOH Add 15 mL water to flask Add 5.2 mL 50% NaOH (19.4 N) to 25 ml volumetric flask Mix Cool to room temperature Bring volume to 25 mL with water Store in a plastic bottle at room temperature Add 4 g NaOH pellets to 25 ml volumetric flask Add 20 mL water volumetric flask Mix until dissolved Cool to room temperature Bring volume to 25 mL with water Store in a plastic bottle at room temperature Prepare calibrators in a 2 to 1 MPA:Assay Buffer Solution 0 uM GSSG - MPA:Assay Buffer 5 uM GSSG - 25 uL calibrator concentrate in 975 uL MPA:Assay Buffer Solution 10 uM GSSG - 50 uL calibrator concentrate in 950 uL MPA:Assay Buffer Solution Note: Calibrators will be treated and assayed the same as the samples except they don't need to be frozen

If you run separate vials, double the syringe and swab counts and add a second BAC water draw per session

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Sections were permeabilized and blocked in 5% donkey serum with 0.3% Triton in PBS for 2 h at room temperature (RT)

10.1158/0008-5472.CAN-10-2638 48