α-Naphthyl Butyrate Esterase Stain

α-Naphthyl Butyrate Esterase Stain (α-NBE)

Principle of α-Naphthyl Butyrate Esterase Stain (α-NBE)

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.

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).

A. Preparation of Working Solutions

1. 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.

2.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.

B. 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.

Interpreting Results

The presence of red or red-brown granules within the cytoplasm signifies a positive reaction.

Methods of α-Naphthyl Butyrate Esterase Stain (α-NBE):

  • Working solution Preparation (for 1 test only):
  • Device required: dispensable cylinder, micropipette, expendable tips, dropper;
  • Guidance: Add 10µl arrangement B to 10µl arrangement C, blend well and sit tight for 1 moment;
  • Add 4ml arrangement D and 200µl arrangement E. Blend well (Add 2 drops of arrangement NaFfor NaF hindrance test.)

Solution B

Solution C

Solution BD

Solution E
Tube  Α10µl10µl4µl200µl
Tube B
(inhibition test)


  • 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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