1. Aspirate the solution and wash with 350µL of 1× Wash Solution to each well using a squirt bottle,
multi-channel pipette, manifold dispenser or autowasher, and let it sit for 1~2 minutes. Remove the remaining liquid from all wells completely by snapping the plate onto absorbent paper. Totally wash 3 times. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and blot it against absorbent paper.
2. Add 100µL of Detection Reagent B working solution to each well. Incubate for 30 minutes at 37
oC after covering it with the Plate sealer.
6. Repeat the aspiration/wash process for total 5 times as conducted in step 4.
3. Add 90µL of Substrate Solution to each well. Cover with a new Plate sealer. Incubate for 15 - 25 minutes at
37oC (Don't exceed 30 minutes). Protect from light. The liquid will turn blue by the addition of Substrate
Solution.
4. Add 50µL of Stop Solution to each well. The liquid will turn yellow by the addition of Stop solution. Mix the liquid by tapping the side of the plate. If color change does not appear uniform, gently tap the plate to ensure thorough mixing.