NADH NBT Staining

SOP

• SOP - Flammables and Combustibles
• SOP- Irritants

Tris Buffer .2M pH 7.4

1. Warm Tris buffer to 37°C in water bath.
2. Use hot plate to keep the buffer at 37°C, pH the buffer with HCl. (pH changes with temperature)

Methods

1. 20mg NBT aka Nitrotetrazolium Blue Chloride or Nitro Blue Tetrazolium
2. 80mg of NADH aka β-Nicotinamide adenine dinucleotide, reduced disodium salt hydrate
3. 100mL of Tris Buffer
4. 100mL fits in a staining dumpster
5. Incubate for 30 minutes at 37°C

Dehydrate and coverslip

1. Rinse 3-4 times under running DI water
2. Immerse in 50% ethanol for 2x 30 seconds
3. Immerse in 70% ethanol for 2x 30 seconds
4. Immerse in in 95% ethanol for 2x 30 seconds
5. Immerse in in 100% ethanol for 2x 30 seconds
6. Immerse in 1:1 xylene ethanol for 2 minutes for 2x 30 seconds
7. Repeat twice more using fresh xylene. Check staining under the microscope
8. Coverslip using Permount