Alkaline Phosphatase-85

Reagent Preparation

Fast Blue, Fast Violet, Naphthol AS-MX, and Mayer’s Hematoxylin are all provided ready to use. Prepare working Citrate solution by diluting 2 mL Citrate concentrate to 100 mL deionized water. Prepare fixative by warming working Citrate solution to room temperature. Stir and add 2 volumes of Citrate solution to 3 volumes of acetone. Use once then discard.

Procedure

  1. Dissolve contents of a Fast Blue RR Salt capsule or a Fast Violet B capsule in a Coplin jar containing 48 mL of room temperature deionized water.
  2. Add 2 mL Naphthol AS-MX Phosphate Alkaline solution to the Coplin jar. Mix.
  3. Fix slides in Citrate fixative for 30 seconds at room temperature.
  4. Rinse gently in deionized water for 45 seconds.
  5. Add slides to alkaline-dye mixture (step 2) and incubate for 30 minutes protected from direct light.
  6. Rinse thoroughly in deionized water. Do not allow to air dry.
  7. Stain in Mayer’s Hematoxylin solution for 10 minutes.
  8. If using Fast Blue RR Salt, rinse slides for 3 minutes in deionized water. If using Fast Violet B Salt rinse with alkaline tap water or Scott’s Tap Water substitute.

Results

Sites of alkaline phosphatase activity will appear as blue or red granular staining.

See product insert sheet for method of scoring stain intensity.

Alkaline Phosphatase-85

Reagents

     
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