Histology

scientificprotocols authored almost 4 years ago

Method:

  1. Incubate sections in the incubating medium at 37°C for 5 - 12 hours. Long incubation periods are needed to get significantly visible reaction product.
  2. Wash in distilled water for 2 minutes.
  3. Counterstain with Methyl Green for 5 minutes.
  4. Wash in distilled water for 2 minutes.
  5. Air dry and coverslip.
  6. Results:
    • Nuclei - dark green
    • Cytoplasm - light green
    • Sites of enzyme activity - red

Solutions:

  1. Incubating Medium
    • Combine 20 ml of buffer solution, 48 ml of distilled water and 4 ml. of substrate solution.
    • Combine 3.2 ml of pararosaniline solution with 3.2 ml of sodium nitrite solution. Mix for 1 minute.
    • Add the second solution to the first
    • Adjust pH to 5.
  2. Buffer Solution
    • Anhydrous sodium acetate - 5.9 g
    • Sodium barbiturate - 14.7 g
    • Distilled water (boiled) - 500 ml
    • Do not adjust the pH of the buffer and store at 4°C.
  3. Substrate Solution
    • Naphtol AS-BI phosphatease, sodium salt (Sigma) - 40 mg
    • N.N-dimethylformamide - 4 ml
  4. Pararosaniline Solution
    • Pararosaniline (C.I.# 42500) - 2 g
    • 2N HCl - 50 ml
    • Use heat to dissolve, filter when cool and store at 4°C.
  5. Sodium Nitrite Solution
    • Sodium Nitrite - 1 gm
    • Distilled Water - 25 ml
    • Prepare fresh and store at 4°C.
  6. Methyl Green
    • Methyl Green (C.I.# 42585) - 1 g
    • Phosphate/citrate buffer 0.1M pH 4.0 - 100 ml

DOI

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