Enzyme Histochemistry Staining

By /

Introduction

  • Enzyme histochemistry staining is used to localise and demonstrate enzyme activity directly within tissues and cells.
  • Unlike routine histological stains, it provides information about the functional and metabolic status of tissues.
  • The technique is based on the reaction between a specific enzyme and its substrate, producing a visible colored reaction product.
  • In muscle pathology, ATPase staining helps differentiate type I (slow-twitch) and type II (fast-twitch) muscle fibers.
  • NADH-tetrazolium reductase (NADH-TR) staining demonstrates the internal architecture and oxidative enzyme activity of muscle fibers.
  • Enzyme histochemistry can reveal structural abnormalities that are not visible with routine H&E staining.
  • It is valuable for diagnosing metabolic muscle disorders by demonstrating absent or reduced enzyme activity, such as myophosphorylase deficiency in McArdle Disease.
  • Enzyme histochemistry remains an important diagnostic tool in neuromuscular pathology despite advances in immunohistochemistry and molecular techniques.

Adenosine Triphosphatase Stain

Principle

  • Myofibrillar ATPase is an enzyme associated with the contractile proteins of skeletal muscle.
  • The activity of this enzyme varies among different muscle fiber types and is influenced by pH.
  • Following incubation with ATP, inorganic phosphate is released.
  • The phosphate reacts with cobalt chloride to form cobalt phosphate, which is subsequently converted into black cobalt sulfide by ammonium sulfide.
  • The intensity of staining reflects ATPase activity and allows differentiation of muscle fiber types.
  • By using different pre-incubation pH conditions (9.4, 4.6, and 4.3), specific muscle fiber populations can be identified.

Sections

  • Fresh unfixed cryostat sections
  • Thickness: 8–10 μm

Preparation of Staining Solutions

1. 0.1 M Glycine Buffer

Reagent Quantity
Glycine 0.75 g
Sodium Chloride (NaCl) 0.585 g
Distilled Water To make 100 ml

2. 0.1 M Glycine Buffer with 0.75 M Calcium Chloride (pH 9.4)

Reagent Quantity
0.1 M Glycine Buffer 50 ml
0.75 M Calcium Chloride Solution 10 ml

Adjust pH to 9.4 using approximately 22 ml of 0.1 M NaOH.

3. Sodium Acetate Buffer

  • 0.1 M Sodium Acetate Buffer
  • 10 mM EDTA
  • pH 4.3
  • pH 4.6

Used for pre-incubation.

4. Incubating Solution

Reagent Quantity
ATP 5 mg
0.1 M Glycine Buffer with 0.75 M CaCl₂ 10 ml

Adjust pH to 9.4.

Staining Procedure at pH 9.4

1. Incubation

  • Incubate sections in incubating solution.
  • 30 minutes at 37°C.

2. Washing

  • Rinse thoroughly in distilled water.

3. Cobalt Chloride Treatment

  • Immerse in 2% cobalt chloride for 5 minutes.

4. Washing

  • Wash in tap water.
  • Follow with three changes of distilled water.

5. Ammonium Sulfide Treatment

  • Immerse in diluted (1:10) ammonium sulfide solution for 30 seconds.
  • Perform inside a fume cupboard.

6. Washing

  • Wash thoroughly in running tap water.

7. Counterstaining (Optional)

  • Lightly stain with Harris hematoxylin.
  • Blue in tap water.

8. Mounting

  • Mount in aqueous mountant or
  • Dehydrate, clear, and mount in DPX.

Staining procedure at pH 4.3 and pH 4.6

1. Pre-incubation

  • Incubate freshly cut sections at 4°C.
  • Use appropriate sodium acetate buffer (pH 4.3 or 4.6).
  • Duration: 10 minutes.

2. Washing

  • Rinse briefly in distilled water.

3. Continue Procedure

  • Proceed from Step 1 of the pH 9.4 method.

Results

ATPase Reaction Pattern

Fiber Type pH 9.4 pH 4.6 pH 4.3
Type I White Black Black
Type IIA Black/Intermediate White White
Type IIB Black Intermediate White
Type IIC Black Black Black/Intermediate

NADH-TR Stain

Nicotinamide Adenine Dinucleotide Dehydrogenase–Tetrazolium Reductase (NADH-TR) Stain

Principle

  • NADH serves as an electron donor and is oxidized by mitochondrial and sarcoplasmic oxidative enzymes.
  • The released electrons reduce Nitro Blue Tetrazolium (NBT) to an insoluble blue-purple formazan precipitate.
  • The amount of formazan deposited is proportional to oxidative enzyme activity.
  • Therefore, fibers rich in mitochondria stain more intensely than fibers with lower oxidative capacity.

Enzyme Demonstrated

  • NADH Dehydrogenase
  • Tetrazolium Reductase Activity
  • Oxidative Enzyme Systems

Sections

  • Fresh unfixed cryostat sections
  • Thickness: 8–10 μm

Preparation of staining Solutions

1. Nitro Blue Tetrazolium (NBT) Stock Solution

Reagent Quantity
Nitro Blue Tetrazolium 20 mg
Distilled Water 0

Storage

  • Store in aliquots at −20°C.

2. NADH Stock Solution

Reagent Quantity
NBT Stock Solution 6.25 ml
0.2 M Tris Buffer (pH 7.4) 1.25 ml
0.5 M Cobalt Chloride 1.25 ml
Distilled Water 8.75 ml

Storage

  • Store in aliquots at −20°C.

3. Incubating Solution

Reagent Quantity
NADH Stock Solution 1 ml
NADH 1 mg

Prepare fresh before use.

Staining procedure 

1. Incubation

  • Incubate sections in incubating solution.
  • Temperature: 37°C
  • Duration: 30 minutes

2. Fixation

  • Drain excess incubating solution.
  • Transfer sections directly into:
    • 10% formalin in tap water
  • Fix for 15 minutes.

3. Washing

  • Wash thoroughly in tap water.

4. Dehydration

  • Pass through graded alcohols.
  • Clear in xylene.

5. Mounting

  • Mount in DPX.

Results

Reaction Product

  • Blue-grey formazan deposit

Staining Intensity of Muscle Fibers

Fiber Type Staining Intensity
Type I Strong
Type IIA Intermediate
Type IIB Weak
Mitochondrial Aggregates Very Strong

Succinate Dehydrogenase Stain

Clinical Significance

SDH staining is useful for:

  • Evaluation of mitochondrial content.
  • Diagnosis of mitochondrial myopathies.
  • Assessment of muscle fiber oxidative capacity.
  • Identification of fiber-type distribution.
  • Detection of mitochondrial proliferation and aggregates.
  • Investigation of congenital and metabolic myopathies.

Principle

  • Succinate dehydrogenase catalyzes the oxidation of succinate to fumarate in the Krebs cycle.
  • During this reaction, electrons are transferred to Nitro Blue Tetrazolium (NBT), reducing it to an insoluble blue-purple formazan precipitate.
  • The amount of formazan deposited is proportional to SDH activity and mitochondrial content.
  • Fibers with greater oxidative capacity therefore stain more intensely.

Enzyme Demonstrated

  • Succinate Dehydrogenase (SDH)
  • Mitochondrial oxidative enzyme activity
  • Complex II of the Electron Transport Chain

Sections

  • Fresh unfixed cryostat sections
  • Thickness: 8–10 μm

Preparation of Staining Solutions

1. 0.2 M Phosphate Buffer

Solution A

Reagent Quantity
Potassium Dihydrogen Orthophosphate 2.72 g
Distilled Water 100 ml

Solution B

Reagent Quantity
Disodium Hydrogen Orthophosphate 0.57 g
Distilled Water 20 ml

Working Phosphate Buffer

Reagent Quantity
Solution A 4 ml
Solution B 16 ml

2. 0.2 M Sodium Succinate Solution

Reagent Quantity
Sodium Succinate 1.08 g
Distilled Water 20 ml

3. SDH Incubating Solution

Reagent Quantity
0.2 M Phosphate Buffer 15 ml
0.2 M Sodium Succinate 15 ml
Nitro Blue Tetrazolium (NBT) 30 mg

Adjust pH to 7.3–7.6.

Storage

  • May be stored in aliquots at −20°C.

Staining Procedure

1. Incubation

  • Incubate sections in SDH incubating solution.
  • Temperature: 37°C
  • Duration: 1–2 hours

2. Fixation

  • Drain sections.
  • Transfer directly into:
    • 10% formalin in tap water
  • Fix for 15 minutes.

3. Washing

  • Wash thoroughly in tap water.

4. Dehydration

  • Pass through graded alcohols.
  • Clear in xylene.

5. Mounting

  • Mount in DPX.

Results

Reaction Product

  • Blue-grey formazan deposit

Staining Pattern

Fiber Type Appearance Activity
Type I Dark Blue-Grey High
Type IIA Pale Blue-Grey Intermediate
Type IIB Weak Blue-Grey Low
Mitochondrial Aggregates Very Dark Blue-Grey Very High

Cytochrome Oxidase Stain

Clinical Significance

COX staining is useful for:

  • Diagnosis of mitochondrial myopathies.
  • Detection of respiratory chain defects.
  • Identification of COX-deficient fibers.
  • Assessment of mitochondrial function.
  • Investigation of neuromuscular disorders.
  • Evaluation of mitochondrial DNA-related diseases.

Principle

  • Cytochrome c oxidase catalyzes the transfer of electrons from reduced cytochrome c to molecular oxygen in the final step of the electron transport chain.
  • During incubation, cytochrome oxidase oxidizes cytochrome c, and the reaction results in oxidation of 3,3′-Diaminobenzidine (DAB).
  • The oxidized DAB polymerizes and forms an insoluble brown reaction product at the sites of enzyme activity.
  • The intensity of the brown staining reflects the level of cytochrome oxidase activity within mitochondria.

Enzyme Demonstrated

  • Cytochrome c Oxidase (COX)
  • Mitochondrial Complex IV
  • Oxidative Phosphorylation Activity

Sections

  • Fresh unfixed cryostat sections
  • Thickness: 8–10 μm

Preparation of Incubating Solution

Reagent Quantity
Catalase (20 μg/ml) 1 ml
Cytochrome c (Type II) 10 mg
0.1 M Phosphate Buffer (pH 7.4) 9 ml
DAB (3,3′-Diaminobenzidine tetrahydrochloride) 5 mg

Preparation of Catalase Solution

  • Dissolve 4 mg catalase in 10 ml distilled water.
  • Remove 2.5 ml.
  • Make up to 50 ml with distilled water.

pH

  • Adjust to pH 7.4 using:
    • 0.1 M NaOH or
    • 0.1 M HCl

Storage

  • May be stored in aliquots at −20°C.

Staining Procedure

1. Incubation

  • Incubate sections in incubating solution.
  • Temperature: 37°C
  • Duration: 2–3 hours

2. Washing

  • Rinse thoroughly in distilled water.

3. Fixation

  • Fix in formal calcium for 15 minutes.

4. Washing and Bluing

  • Wash thoroughly.
  • Blue if required.

5. Dehydration

  • Pass through graded alcohols.
  • Clear in xylene.

6. Mounting

  • Mount in DPX.

Results

Staining Pattern

Structure Appearance
COX-positive fibers Brown
COX-deficient fibers Pale or Unstained
Areas of high mitochondrial activity Dark Brown

Alkaline Phosphatase Stain

Clinical Significance

Alkaline phosphatase staining is useful for:

  • Evaluation of inflammatory myopathies.
  • Demonstration of muscle regeneration.
  • Identification of connective tissue proliferation.
  • Assessment of muscle fiber injury.
  • Investigation of certain neuromuscular disorders.

Principle

  • Alkaline phosphatase hydrolyzes α-naphthyl phosphate to release α-naphthol.
  • The liberated α-naphthol immediately reacts with the diazonium salt Fast Red TR, producing an insoluble azo dye precipitate at the site of enzyme activity.
  • The resulting reddish-brown reaction product indicates the location and intensity of alkaline phosphatase activity within the tissue.

Enzyme Demonstrated

  • Alkaline Phosphatase (ALP)

Fixation

Recommended Fixatives

  • Formal calcium at 4°C
  • Formal vapor fixation

These fixation methods preserve alkaline phosphatase activity.

Sections

  • Pre-fixed cryostat sections (preferred)
  • Thickness: 8–10 μm

Preparation of Incubating Medium

Reagent Quantity
Sodium α-Naphthyl Phosphate 10 mg
0.2 M Tris Buffer (pH 10.0) 10 ml
Fast Red TR (Diazonium Salt) 10 mg

Preparation

  1. Dissolve sodium α-naphthyl phosphate in Tris buffer.
  2. Add Fast Red TR.
  3. Mix thoroughly.
  4. Filter immediately before use.

Final pH

  • Between 9.0 and 9.4

The solution should be used fresh.

Staining Procedure

1. Incubation

  • Bring fixed sections to water.
  • Incubate in freshly prepared incubating medium.
  • Room temperature.
  • Duration: 10–60 minutes.

2. Distilled Water Wash

  • Wash for 3 minutes in distilled water.

3. Acetic Acid Wash

  • Wash in 1% acetic acid for 2 minutes.

This stops the enzymatic reaction and removes excess dye.

4. Final Rinse

  • Rinse thoroughly in distilled water.

5. Mounting

  • Mount in aqueous mounting medium.

Results

Structure Colour
Alkaline Phosphatase Activity Reddish-Brown

Acid Phosphatase Stain

Clinical Significance

Acid phosphatase staining is useful for:

  • Diagnosis of lysosomal storage disorders.
  • Detection of vacuolar myopathies.
  • Assessment of muscle fiber degeneration.
  • Demonstration of autophagic activity.
  • Evaluation of inflammatory muscle diseases.
  • Identification of lysosome-rich cells.

Principle

  • Acid phosphatase hydrolyzes Naphthol AS-BI phosphate to release free naphthol.
  • The liberated naphthol immediately couples with a diazonium compound generated from sodium nitrite and pararosaniline hydrochloride, forming an insoluble red azo dye at the site of enzyme activity.
  • The intensity of the red reaction product reflects the level of acid phosphatase activity within the tissue.

Enzyme Demonstrated

  • Acid Phosphatase (ACP)
  • Lysosomal enzyme activity

Fixation

Recommended Fixatives

  • Formal calcium at 4°C
  • Formal vapor fixation

These methods preserve enzyme activity while maintaining tissue morphology.

Sections

  • Pre-fixed cryostat sections (preferred)
  • Unfixed cryostat sections may also be used
  • Thickness: 8–10 μm

Preparation of Solutions

1. Substrate Solution (A)

Reagent Quantity
Naphthol AS-BI Phosphate 10 mg
Dimethyl Formamide 1 ml

2. Buffer Solution (B)

Reagent Quantity
Sodium Acetate (3H₂O) 1.94 g
Sodium Barbitone 2.94 g
Distilled Water 100 ml

3. Sodium Nitrite Solution (C)

Reagent Quantity
Sodium Nitrite 400 mg
Distilled Water 10 ml

4. Pararosaniline Hydrochloride Stock Solution (D)

Reagent Quantity
Pararosaniline Hydrochloride 1 g
Distilled Water 20 ml
Concentrated Hydrochloric Acid 5 ml

Preparation

  • Heat gently until dissolved.
  • Allow to cool.
  • Filter before use.

Preparation of Incubating Solution

Reagent Quantity
Solution A 0.5 ml
Solution B 2.5 ml
Solution C 0.4 ml
Solution D 0.4 ml
Distilled Water 6 ml

Critical Step

For successful staining:

  • Mix equal volumes of Solution C and Solution D first.
  • Allow to stand for 2 minutes.
  • Then add to Solutions A and B.

Final pH

  • Between 4.7 and 5.0
  • Adjust if necessary using 0.1 M NaOH.

Staining Procedure

1. Incubation

  • Incubate sections in freshly prepared incubating solution.
  • Temperature: 37°C
  • Duration: 15–60 minutes

2. Washing

  • Wash thoroughly in distilled water.

3. Counterstaining

  • Counterstain with hematoxylin.

4. Bluing

  • Wash in running tap water.

5. Mounting

Either:

  • Mount in aqueous mounting medium

or

  • Rapidly dehydrate through fresh alcohols
  • Clear in xylene
  • Mount in DPX

Results

Structure Colour
Acid Phosphatase Activity Red
Nuclei Blue

Phosphofructokinase Stain

Clinical Significance

PFK staining is useful for:

  • Diagnosis of glycolytic pathway defects.
  • Investigation of glycogen storage diseases.
  • Assessment of muscle energy metabolism.
  • Evaluation of metabolic myopathies.
  • Detection of phosphofructokinase deficiency.

Principle

  • Phosphofructokinase catalyzes the phosphorylation of fructose-6-phosphate using ATP.
  • Through a series of coupled enzymatic reactions, reducing equivalents are generated, which reduce Nitro Blue Tetrazolium (NBT) to an insoluble blue-purple formazan precipitate.
  • The intensity of the formazan deposit corresponds to the level of phosphofructokinase activity within the tissue.

Enzyme Demonstrated

  • Phosphofructokinase (PFK)
  • Glycolytic enzyme activity

Sections

  • Fresh unfixed cryostat sections
  • Thickness: 8–10 μm

Preparation of PFK Incubating Medium

Reagent Quantity
20 mmol Sodium Arsenate 8.0 ml
10 mmol D-Fructose-6-Phosphate* 3.2 ml
10 mmol Nicotinamide Adenine Dinucleotide (NAD)* 1.6 ml
10 mmol Adenosine Triphosphate (ATP)* 1.6 ml
40 mmol Magnesium Sulphate 0.4 ml
Nitro Blue Tetrazolium (NBT) 6.4 mg
Distilled Water 1.2 ml

Important

The following components must be prepared fresh before use:

  • D-Fructose-6-phosphate
  • NAD
  • ATP

Final pH

  • Adjust to pH 7.0

Staining procedure

1. Incubation

  • Filter the freshly prepared PFK incubating medium.
  • Pre-warm to 37°C.
  • Incubate sections in a moist chamber.
  • Temperature: 37°C
  • Duration: 1–2 hours

2. Washing

  • Rinse thoroughly in distilled water.

3. Acetone Treatment

To remove excess red monoformazan:

  • Immerse briefly in:
    • 30% acetone
    • 60% acetone
    • 90% acetone

Only a few seconds in each solution are required.

4. Dehydration and Mounting

  • Dehydrate.
  • Clear.
  • Mount in Pertex.

Results

Structure Colour
Sites of PFK Activity Blue to Purple
Scroll to Top
Enable Notifications OK No thanks