α-Naphthyl Butyrate Esterase Stain (α-NBE)
Table of Contents
1. Principle of α-Naphthyl Butyrate Esterase Stain
α-Naphthyl Butyrate Esterase Stain, also known as α-NBE stain, is a method used to detect the presence of esterases within cells. Esterases are enzymes that catalyze the hydrolysis of ester bonds. In this staining technique, the α-Naphthyl Butyrate substrate is hydrolyzed by esterases under alkaline conditions, leading to the formation of α-naphthol. This α-naphthol then reacts with a diazonium salt to produce an insoluble colored precipitate within the cytoplasm of the cells. It’s important to note that this stain is termed “Non-specific Esterase Stain” due to its lack of specificity against esterases.
2. Main Components
The staining process involves several key components:
- Fixative: Preserves cell morphology (Formaldehyde).
- Diazotization Solution: Forms colored precipitate (Parafuchsin).
- Sodium Nitrite Solution: Aids in reaction (Sodium nitrite).
- Phosphate Buffer: Maintains pH (Phosphate).
- α-Naphthyl Butyrate Solution: Substrate (α-Naphthyl Butyrate).
- Methyl Green Solution: Enhances contrast (Methyl green).
- NaF Solution: Potential stabilizer (Sodium Fluoride).
3. Preparation of Working Solutions
A. Dip Stain Working Solution:
- Prepare a dye vat with a volume of 50ml.
- Combine solution B and solution C (0.1ml each) and allow them to stand for 2 minutes.
- Introduce 40ml of solution D to the dye vat.
- Pour the diazonium salt solution into the dye vat and ensure thorough mixing.
- Add 2ml of solution E and gently agitate.
- For inhibition tests, introduce 1.3ml of NaF solution.
B. Drops Stain Working Solution:
- Merge 5μl of solution B and 5μl of solution C within a test tube, allowing a 1-minute interval.
- Introduce 2ml of solution D and 100μl of solution E, ensuring comprehensive mixing.
- For inhibition tests, add a single drop of NaF solution.
4. Staining Process
- Fix the cell smear and allow it to rest in solution A for 30 to 60 seconds. Rinse with distilled water and subsequently dry the smear.
- Apply the working solution onto the slide, either through direct application or dipping. Wait 60 minutes at room temperature before incubating.
- Implement a restaining step using solution F for 1 to 2 minutes. Rinse the smear and allow it to air dry, preparing it for microscopic examination.
5. Interpreting Results
The presence of red or red-brown granules within the cytoplasm signifies a positive reaction.
6. Methods OF α-Naphthyl Butyrate Esterase Stain :
The following steps should be followed to prepare a working solution (for test only):
- Device required: expendable cylinder, micropipette, dispensable tips, dropper;
- Guidance: Add 10µl arrangement B to 10µl arrangement C, blend well and hang tight for 1 moment;
- Add 4ml arrangement D and 200µl arrangement E. Blend well (Add 2 drops of arrangement NaFfor NaF restraint test.)
| Solution B | Solution C | Solution D | Solution E | Solution NαF | |
| Tube A | 10µl | 10µl | 4µl | 200µl | – |
| Tube B (inhibition test) | 10µl | 10µl | 4µl | 200µl | 2drops |
7. Precautions
- Ensure that the stain is used on appropriate slides and in the recommended staining methods (drip staining or dip staining) based on the provided quantities.
- Thoroughly mix solutions B and C to ensure a consistent and effective staining process.
- Employ fresh specimens to maintain the positivity rate and avoid false negatives.
- Prepare the working solution shortly before staining and use it within 10 minutes to ensure optimal results.
- Seal reagent bottles tightly after use to prevent evaporation and maintain the quality of the reagents.
- This staining kit should be utilized by professionals with a clear understanding of its application and result interpretation.
- Carefully read the instruction manual before usage, follow guidelines on expiration dates, and prioritize personal hygiene during handling.
- The package should include production lot numbers and expiration dates for quality control.
- Properly dispose of waste following hospital or environmental protection regulations.
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α-NBE staining offers insights into cellular enzymatic activity. By understanding its principles and following precautions, researchers can utilize this technique effectively.