Acid α-Naphthyl Acetate Esterase (ANAE) Staining, also known as non-specific esterase staining, is based on the principle that under acidic conditions, acid esterase in cells hydrolyzes α-naphthyl acetate to produce α-naphthol. The α-naphthol then couples with hexazotized pararosaniline to form an insoluble red precipitate, localized in the cytoplasm.
Neutral Red Hydrochloride Solution (4%) is prepared from hydrochloric acid and new fuchsin, among other components. When mixed in equal proportions with a 4% nitrite solution, it produces hexazotized pararosaniline (a diazonium salt), which can be used for Acid α-Naphthyl Acetate Esterase (ANAE) staining. This reagent is intended for scientific research only and is not suitable for clinical diagnosis or other purposes.
Reagents, consumables and Equipments not provided
- Microscope, glass slides, wet box,
- 4% nitrite solution, and other necessary supporting reagents for the experiment.
Operating Procedure (for reference only)
1. Fix blood, bone marrow, or cell smears, or frozen sections with formaldehyde-acetone buffer for 30–60 seconds. Rinse with water for 5 minutes.
2. Prepare the relevant staining incubation solution and proceed according to the operational workflow. Alternatively, refer to the Acid α-Naphthyl Acetate Esterase Staining Solution (ANAE method) for guidance.
Staining Results
Positive areas appear dark red or brown.
Precautions
1. Blood or bone marrow cell smears should be fresh and of appropriate thickness. Staining is generally recommended within 2 days to avoid affecting enzyme activity.
2. Hydrochloric acid pararosaniline solution contains hydrochloric acid and is somewhat corrosive. Handle with care.
3. Hydrochloric acid pararosaniline solution and nitrite solution should be mixed in equal proportions immediately before use. Wait for 1 minute before proceeding with further steps. Note that mixing these solutions produces irritating gases.
4. For safety and health, wear a lab coat and disposable gloves during operation.
5. After opening the reagent, use it as soon as possible to prevent affecting subsequent experimental results.
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