NADPH Stain

1. set small oven to 37^ºC (setting 1)

2. Prepare TBS (pH 7.3 and 8.0)

3. Organize trays and label

4. Measure 19.84 ml TBS (pH8.0) into small flask (label)

5. Pipette (tip under solution) 160ul triton-x into flask very slowly

a. First 100ul ® 60ul

6. Slowly magnetic stir throughout as not to introduce oxygen to the solution (be sure the plate is not heated)

7. Weigh 24 mg C₄H₆O₅ (malic acid), add to solution

! Next step is light sensitive – turn off main lights and wear gloves !

8. Nitro Blue Tetrazolium (NBT)

a. measure 20 mg

b. wrap flask with Al foil

c. add NBT

9. Weigh 8mg NADPH and add to solution

ü ensure homogenous solution

This produces the NADPH stain

10. Rinse tissue in TBS (pH 7.3) 2x for ~1 min.

11. Pour stain into each well evenly – transfer tissue from

TBS into NADPH wells

12. Put in oven ~20 min.

13. 2 – 1 min. rinses in TBS (pH 7.3)

14. Mount ASAP (if stored, keep in TBS)

15. Dispose of waste in Toxic waste bottle

Clean all instruments