Nucleic acid, DNA and RNA Stains

Introduction

• Nucleic acids are the genetic materials of the cell and occur as DNA and RNA.
• DNA is primarily located in the nucleus, whereas RNA is found in the nucleus, nucleolus, ribosomes, and cytoplasm.
• Routine H&E staining cannot specifically differentiate DNA from RNA.
• Special histochemical stains are used to selectively demonstrate and localize nucleic acids within cells.
• The Feulgen reaction is the classical histochemical stain specific for DNA.
• Methyl Green–Pyronin stain differentiates DNA and RNA in the same tissue section.
• Nucleic acid stains are useful in studying cell proliferation, protein synthesis, and neoplastic transformation.
• These techniques play an important role in histopathology, cytology, molecular biology, and biomedical research.

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Nucleic acids Staining

Hematoxylin and Eosin (H&E) Stain

Introduction

• Hematoxylin and Eosin (H&E) stain is the most widely used and important staining technique in histopathology.
• It is considered the gold standard routine stain for the microscopic examination of tissues and serves as the foundation for tissue diagnosis in pathology laboratories worldwide.
• The H&E stain provides excellent contrast between the nucleus and cytoplasm, allowing clear visualization of cellular and tissue architecture.
• Hematoxylin stains nuclei and other basophilic structures blue to purple, while eosin stains cytoplasm and extracellular components in varying shades of pink to red.
• This combination produces a comprehensive overview of tissue morphology and enables the identification of both normal and pathological changes.

Principle

• The H&E stain is based on the interaction of acidic and basic dyes with tissue components.

Hematoxylin

• Hematoxylin itself is not a dye. After oxidation to hematein and combination with a mordant (usually aluminum), it behaves as a basic dye and binds to acidic tissue components.

Structures stained by hematoxylin:

• Nuclei
• Chromatin
• Nucleoli
• Ribosomes
• Rough endoplasmic reticulum

Eosin

• Eosin is an acidic dye that binds to basic proteins within tissues.

Structures stained by eosin:

• Cytoplasm
• Muscle fibers
• Collagen
• Connective tissue
• Red blood cells

 

Fixation

Common fixatives include:

• 10% Neutral Buffered Formalin (preferred)
• Bouin’s fixative
• Zenker’s fixative
• Alcohol-based fixatives

Sections

• Paraffin sections (3–5 μm)
• Frozen sections
• Cytological preparations

Reagents Required

• Xylene
• Absolute alcohol
• 95% alcohol
• 70% alcohol
• Distilled water
• Harris’ or Mayer’s hematoxylin
• 1% Acid alcohol
• Scott’s tap water substitute (or ammonia water)
• Eosin Y solution
• DPX mountant

Staining Procedure

1. Deparaffinization

Purpose: Removes paraffin wax from tissue sections.

2. Hydration

Purpose: Gradually rehydrates the tissue.

3. Nuclear Staining

Purpose: Stains nuclei blue-purple.

4. Washing

• Wash in running tap water for 5 minutes.

Purpose: Removes excess hematoxylin.

5. Differentiation

Purpose: Removes excess hematoxylin from non-nuclear structures.

6. Washing

• Wash thoroughly in tap water.

7. Bluing

Purpose: Converts hematoxylin to a stable blue color.

8. Washing

• Wash in running tap water for 2–3 minutes.

9. Counterstaining

Purpose: Stains cytoplasm and connective tissue pink.

10. Dehydration

Purpose: Removes water from the section.

11. Clearing

Purpose: Makes tissue transparent and prepares for mounting.

12. Mounting

• Mount with DPX and cover with a coverslip.

Results

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Deoxyribonucleic acid (DNA) Staining

Feulgen Nuclear Reaction for DNA

Introduction

• The Feulgen reaction is the classical and most specific histochemical method for demonstrating DNA (Deoxyribonucleic Acid) in tissue sections and cells.
• Developed by Robert Feulgen and Heinrich Rossenbeck in 1924, this technique selectively stains DNA without reacting with RNA, making it a highly specific method for nuclear DNA identification.

Clinical Significance

The Feulgen reaction is useful for:

• Demonstration of nuclear DNA.
• Study of chromosomes and mitotic figures.
• DNA quantification.
• Evaluation of cell proliferation.
• Detection of nuclear abnormalities.
• Cytogenetic and cancer research.

Principle

• The Feulgen reaction is based on the selective hydrolysis of DNA by hydrochloric acid.
• Controlled acid hydrolysis removes purine bases from DNA, exposing aldehyde groups on the deoxyribose sugar.
• These newly formed aldehyde groups react with Schiff’s reagent, producing a characteristic red-purple (magenta) color at sites containing DNA.
• Since RNA does not undergo this reaction under the same conditions, the stain is considered highly specific for DNA.

Substance Demonstrated

• DNA (Deoxyribonucleic Acid)

Fixation

Suitable Fixatives

• Formalin
• Alcohol-based fixatives
• Carnoy’s fixative

Avoid

• Bouin’s fixative

Preparation of Solutions

1. 1 M Hydrochloric Acid

2. Schiff’s Reagent

Prepared according to standard Schiff reagent protocol.

3. Bisulfite Solution

Prepare fresh when required.

Staining Procedure

1. Hydration

• Bring sections to water.

2. Initial Acid Rinse

• Rinse sections in 1 M HCl at room temperature.

3. Hydrolysis

• Place sections in 1 M HCl at 60°C.

Purpose

• Hydrolyzes DNA.
• Exposes aldehyde groups.

4. Acid Rinse

• Rinse in 1 M HCl at room temperature for 1 minute.

5. Schiff’s Reagent

• Transfer sections to Schiff’s reagent.
• Stain for 45 minutes.

6. Bisulfite Wash

• Rinse in bisulfite solution for 2 minutes.

7. Repeat Bisulfite Wash

• Repeat for another 2 minutes.

8. Third Bisulfite Wash

• Repeat again for 2 minutes.

9. Water Wash

• Wash thoroughly in distilled water.

10. Counterstaining (Optional)

• Counterstain in 1% Light Green for 2 minutes.

11. Wash

• Wash in water.

12. Dehydration and Mounting

• Dehydrate through graded alcohols.
• Clear in xylene.
• Mount in DPX.

Results

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Methyl Green–Pyronin stain

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Enzyme Extraction of DNA Method

Introduction

• The enzyme extraction of DNA method, introduced by Brachet in 1940, is a histochemical control technique used to confirm the specificity of DNA staining methods, particularly the Feulgen reaction.
• The method employs deoxyribonuclease (DNase), an enzyme that selectively digests DNA, thereby removing DNA from tissue sections before staining.

Principle

Deoxyribonuclease hydrolyzes DNA into smaller soluble fragments, which are subsequently removed from the tissue section during washing.

When the Feulgen reaction is performed after digestion:

• The test section lacks DNA and therefore does not stain.
• The control section retains DNA and shows the normal Feulgen-positive reaction.

This demonstrates that the Feulgen reaction is specific for DNA.

Substance Demonstrated

• DNA (by selective removal)

Fixation

Suitable Fixatives

• Formalin
• Alcohol-based fixatives
• Carnoy’s fixative

Avoid

• Potassium dichromate-containing fixatives

Reason: Potassium dichromate inhibits DNase activity.

Sections

• Paraffin sections
• Cryostat sections

Preparation of Digestion Solution

Prepare fresh before use.

Staining procedure

1. Hydration

• Bring both test and control sections to water.

2. Enzyme Digestion

Test Section

• Place in DNase digestion solution.

Control Section

• Place in 0.2 M Tris buffer (pH 7.6).

Incubation

• Temperature: 37°C
• Duration: 4 hours

3. Washing

• Wash thoroughly in running tap water.

4. Feulgen Staining

• Stain both sections using the Feulgen method.

Results

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Ribonucleic acid Staining

Enzyme Extraction of RNA Method

Introduction

• The enzyme extraction of RNA method, developed by Brachet in 1940, is a histochemical control technique used to confirm the specificity of RNA staining methods, particularly the Methyl Green–Pyronin stain.
• The technique employs ribonuclease (RNase), an enzyme that selectively digests RNA while leaving DNA unaffected.

Principle

Ribonuclease hydrolyzes RNA into soluble nucleotide fragments that are subsequently removed from the tissue during washing.

After digestion, the Methyl Green–Pyronin stain is applied:

• RNA is removed and therefore fails to stain red.
• DNA remains intact and stains green.

Comparison of the test and control sections confirms the specificity of RNA staining.

Substance Demonstrated

• RNA (by selective removal)
• DNA remains unaffected

Fixation

Suitable Fixatives

• Carnoy’s fixative
• Formalin

Avoid

• Potassium dichromate-containing fixatives
• Mercuric chloride-containing fixatives

Reason: These inhibit RNase activity and prevent complete RNA digestion.

Sections

• Paraffin sections
• Cryostat sections

Preparation of Digestion Solution

Prepare fresh before use.

Staining procedure

1. Hydration

• Bring both test and control sections to water.

2. Enzyme Digestion

Test Section

• Place in RNase solution.

Control Section

• Place in distilled water.

Incubation

• Temperature: 37°C
• Duration: 1 hour

3. Washing

• Wash thoroughly in distilled water.

4. Methyl Green–Pyronin Staining

• Apply the Methyl Green–Pyronin method to both sections.

Results