NADH NBT Staining

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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