The document discusses routine hematoxylin and eosin (H&E) staining procedures and the use of Mallory’s phosphotungstic acid hematoxylin (PTAH) for histological studies. It details the steps involved in both staining methods, types of hematoxylins, and the specific applications of PTAH in muscle and neuropathology. Proper techniques and troubleshooting tips are also provided to ensure effective staining and accurate analysis.

1. Routine Staining Procedures
Hematoxylin and Eosin Staining, Various
Types of Hematoxylins, and Mallory’s
Phosphotungstic Acid Hematoxylin (PTAH)
Author; Jagroop Singh
PhD Biochemistry

2. Introduction
• Routine staining procedures are essential for
visualizing tissue structure and morphology in
histological studies.
• Hematoxylin and Eosin (H&E) staining is the most
widely used technique, providing excellent
contrast between the nucleus and cytoplasm.
• Mallory’s Phosphotungstic Acid Hematoxylin
(PTAH) is used for demonstrating specific tissue
components, such as muscle striations and fibrin.

3. Hematoxylin and Eosin (H&E) Staining
• Hematoxylin stains the nuclei of cells blue
or purple, providing excellent contrast.
• Eosin stains the cytoplasm and
extracellular matrix pink, complementing
the nuclear stain.
• The H&E staining procedure involves
several steps: fixation, dehydration,
clearing, embedding, sectioning, staining,
and mounting.

4. • H&E Staining Procedure
• 1. **Deparaffinization**:
• - Immerse slides in xylene to remove paraffin wax.
• 2. **Hydration**:
• - Pass slides through descending grades of alcohol to water.
• 3. **Hematoxylin Staining**:
• - Stain slides in hematoxylin solution for 5-10 minutes.
• 4. **Differentiation**:
• - Dip slides in acid alcohol to remove excess hematoxylin.
• 5. **Bluing**:
• - Rinse slides in bluing agent (e.g., ammonia water) to enhance nuclear
staining.

5. • H&E Staining Procedure (Continued)
6. **Eosin Staining**:
- Stain slides in eosin solution for 1-2 minutes.
7. **Dehydration**:
- Pass slides through ascending grades of alcohol to
remove water.
8. **Clearing**:
- Clear slides in xylene to prepare for mounting.
9. **Mounting**:
- Apply mounting medium and coverslip to preserve stained
tissue.

6. Important Considerations
• 1. Ensure complete deparaffinization for
effective staining.
• 2. Adjust hematoxylin and eosin staining times
based on tissue type and desired contrast.
• 3. Thoroughly rinse slides between each step
to prevent contamination.
• 4. Use fresh reagents and solutions for
consistent results.
• 5. Verify dehydration and clearing are
complete before mounting.

7. Types of Hematoxylins
• 1. Mayer's Hematoxylin: A progressive stain used
for nuclear staining without differentiation.
• 2. Harris Hematoxylin: A regressive stain often used
with an acid alcohol differentiation step.
• 3. Gill's Hematoxylin: Available in three strengths,
suitable for both progressive and regressive
staining.
• 4. Ehrlich's Hematoxylin: A naturally ripened stain
providing intense nuclear staining.
• 5. Delafield's Hematoxylin: A ripened stain providing
good nuclear detail.

8. Mallory’s Phosphotungstic Acid Hematoxylin
(PTAH)
• PTAH is used to demonstrate muscle
striations, fibrin, and glial fibers.
• The stain uses phosphotungstic acid to
bind tissue elements, producing a blue-
black color for structures like muscle and a
reddish-brown color for collagen.
• The PTAH technique is valuable in
neuropathology for highlighting astrocytes
and glial cells.

9. • Reagents Required;
• Content:
• Phosphotungstic Acid Hematoxylin (PTAH)
Solution: The main staining agent.
• Potassium Permanganate Solution: Used for
oxidation.
• Oxalic Acid Solution: Used for differentiation.
• Dehydrating Alcohols (70%, 95%, 100%): Used
for dehydration.
• Clearing Agent (e.g., xylene): Used for clearing.
• Mounting Medium and Coverslips: For preserving
stained slides.

10. • PTAH Staining Procedure
• Content:
• 1. Deparaffinization:
• Immerse slides in xylene for 10-15 minutes to
remove paraffin wax.
• Repeat twice to ensure complete removal of wax.
• 2. Hydration:
• Pass slides through descending grades of alcohol
(100%, 95%, 70%) for 5 minutes each to bring slides
to water.
• 3. Potassium Permanganate Oxidation:
• Place slides in 0.25% potassium permanganate
solution for 5 minutes to oxidize tissues.

11. • 4. Oxalic Acid Differentiation:
• Treat slides with 2% oxalic acid for 1-2 minutes until
sections are bleached to a pale yellow.
• 5. Staining with PTAH Solution:
• Stain slides in PTAH solution for 12-24 hours at room
temperature.
• Ensure slides are fully submerged for even staining.
• 6. Rinsing:
• Rinse slides in distilled water for 5 minutes to remove
excess stain.
• 7. Dehydration:
• Pass slides through ascending grades of alcohol (70%,
95%, 100%) for 5 minutes each to remove water.

12. • 8. Clearing:
• Clear slides in xylene for 5 minutes to
prepare for mounting.
• Repeat clearing step twice to ensure
transparency.
• 9. Mounting:
• Apply mounting medium and coverslip to
preserve stained tissue.

13. Applications of PTAH Staining
• 1. Muscle Pathology: Demonstrates cross-
striations and changes in muscle fibers.
• 2. Connective Tissue: Highlights fibrin and
collagen fibers.
• 3. Neuropathology: Identifies glial cells
and structures in the central nervous
system.
• 4. Tumor Pathology: Differentiates
between various tissue components in
tumors.

14. • Troubleshooting Common Issues
• Content:
• Faint Staining:
• Increase staining time or concentration of
PTAH solution.
• Overstaining:
• Reduce PTAH staining time or increase
differentiation time with oxalic acid.
• Incomplete Deparaffinization:
• Ensure sufficient xylene treatment and
extend deparaffinization time if necessary.

15. Conclusion
• Hematoxylin and Eosin (H&E) staining is
fundamental in histology, providing essential
contrast for tissue examination.
• Mallory’s Phosphotungstic Acid Hematoxylin
(PTAH) offers specialized staining for specific
tissue components.
• Understanding the applications and techniques
of these stains is crucial for accurate
histopathological analysis.

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