Principle

The aim of this stain is to demonstrate the presence of granulocytes. Granulocyte lysozomes contain a rather specific hydrolase that can use the Naphtol AS-D Chloroacetate as substrate. The liberated naphtol reacts with the diazonium salt "Fast Red Violet LB", forming red depots.

Reagents

Sigma: Kit num. 91C
Naphtol AS-D Chloroacetate Esterase

Working reagent preparation

1. Mix
• 0,1 ml of the sodium nitrite solution
• 0,1 ml of the Fast Red Violet LB solution
2. Let stand for 2 minutes (diazo preparation)
3. Add 4 ml H2O at 37°C. Mix
4. Add 0,5 ml of Trizmal buffer pH 6,3 . Mix
5. Add 0,1 ml of Naphtol AS-D Chloroacetate reagent. Mix
6. Solution will become slightly red. If the solution forms a precipitate, filter or centrifuge

Staining procedure

1. In a test tube, add 100 à 200 ul of sediment
2. Add to this tube 2 ml of working reagent
3. Incubate at 37°C for 15 minutes
4. Centrifuge and read the results

Nucleus can be stained by adding the methylene blue.

Results

Polynuclear cells stain deep red, while the rest of cells remain unstained, or take a light pink to orange red color due to non-specific adsorbtion. Mastocytes and some macrophages are stained with this method.