2 days ago4 min read
5 days ago4 min read
Mastering the H&E Staining: A Step-by-Step Protocol for Flawless Histology
- CLYTE research team
- 2 days ago
- 4 min read
For over a century, the Hematoxylin and Eosin (H&E) stain has remained the undisputed gold standard in histology and anatomical pathology. It is the cornerstone of tissue-based diagnosis, transforming transparent, paraffin-embedded tissue sections into a vivid map of cellular components.
This simple yet powerful two-dye combination provides a comprehensive view of tissue morphology, allowing pathologists and researchers to identify everything from normal cellular organization to the subtle signs of disease. However, achieving a crisp, high-quality H&E stain requires precision, consistency, and a deep understanding of the entire workflow.
The Core Principle: How H&E Staining Works
The magic of the H&E stain lies in its simple acidic and basic chemistry, which exploits the different chemical properties within a cell.
Hematoxylin (The "H"): Hematoxylin itself is not a dye but, when combined with a metallic "mordant" (like aluminum), it becomes a positively charged, basic complex. This basic dye is attracted to negatively charged, acidic components in the cell, which are known as basophilic. The most prominent basophilic structure is the cell nucleus (due to the nucleic acids, DNA and RNA), which stains a crisp blue or purple.
Eosin (The "E"): Eosin Y is a negatively charged, acidic dye. It is attracted to positively charged, basic components in the tissue, which are known as eosinophilic (or acidophilic). These structures, primarily proteins in the cytoplasm and extracellular matrix (like collagen and muscle fibers), stain in varying shades of pink or red.
This elegant color contrast—blue nuclei against a pink cytoplasm—is what gives histologists a clear, detailed view of the tissue's structure.
Safety and Standardization
Before beginning any staining, laboratory safety and protocol standardization are paramount. All personnel must be aware of the hazards.
Chemical Handling: The H&E protocol involves hazardous chemicals, including flammable solvents like xylene and graded alcohols (ethanol). All deparaffinization and clearing steps involving xylene must be performed inside a certified chemical fume hood.
Personal Protective Equipment (PPE): Always wear a lab coat, nitrile gloves, and safety glasses.
Waste Disposal: Dispose of all solvents and stains according to your institution's hazardous waste guidelines.
Consistency: For research applications, such as those for the AIDS and Cancer Specimen Resource (ACSR), following a precise, documented SOP is critical for ensuring reproducible, high-quality results across all samples.
The Complete H&E Staining Protocol: Step-by-Step
This protocol is a synthesized standard procedure for formalin-fixed, paraffin-embedded (FFPE) tissue sections. Timings may be adjusted based on tissue type, fixative used, and pathologist preference.
1. Deparaffinization and Rehydration
The goal is to remove the paraffin wax and slowly re-introduce water to the tissue.
Xylene I: Immerse slides in xylene (or a xylene substitute) for 5-10 minutes.
Xylene II: Immerse slides in a fresh bath of xylene for 5-10 minutes.
100% Ethanol I: Immerse for 3-5 minutes.
100% Ethanol II: Immerse in a fresh bath for 3-5 minutes.
95% Ethanol: Immerse for 3 minutes.
70% Ethanol: Immerse for 3 minutes.
Running Water Rinse: Rinse slides in a coplin jar under gentle, running tap water for 5 minutes.
2. Hematoxylin Staining (Nuclei)
Stain in Hematoxylin: Immerse slides in a filtered hematoxylin solution (e.g., Mayer's or Harris's) for 3-5 minutes. (Note: some protocols, like Abcam's, suggest 5 minutes, while URMC's suggests 1 minute for their specific reagent).
Water Rinse: Rinse well in tap water until water runs clear.
Differentiation (Regressive Method): Quickly dip slides in acid-alcohol (e.g., 1% HCl in 70% ethanol) for 1-3 seconds. This step is crucial as it removes excess hematoxylin from the cytoplasm, increasing nuclear crispness.
Water Rinse: Immediately rinse in tap water.
Bluing: Immerse slides in a "bluing" solution (e.g., Scott's Tap Water Substitute or ammonia water) for 10-60 seconds. This step changes the hematoxylin from reddish-purple to a sharp blue and is pH-dependent.
Water Rinse: Rinse thoroughly in tap water for 1-5 minutes.
3. Eosin Staining (Cytoplasm)
Eosin Stain: Immerse slides in Eosin Y solution for 30 seconds to 2 minutes. Time depends on the desired intensity.
Water Rinse: Briefly rinse in tap water or distilled water (1-2 dips) to remove excess eosin. Do not rinse excessively, as eosin is water-soluble.
4. Dehydration, Clearing, and Mounting
The goal is to remove all water and prepare the slide for permanent cover-slipping.
95% Ethanol I: Immerse for 2-3 minutes.
95% Ethanol II: Immerse for 2-3 minutes.
100% Ethanol I: Immerse for 2-3 minutes.
100% Ethanol II: Immerse in a fresh bath for 2-3 minutes.
Xylene I: Immerse for 5 minutes.
Xylene II: Immerse in a fresh bath for 5 minutes.
Mounting: Place a drop of permanent mounting medium onto the tissue section and apply a glass coverslip, carefully avoiding air bubbles.
Expected Results
A successful H&E stain will show:
Nuclei: Crisp blue to purple.
Cytoplasm: Varying shades of pink.
Muscle Fibers: Deep pink or red.
Collagen: Pale pink.
Erythrocytes (Red Blood Cells): Bright, intense red or pink-orange.
Best Practices and Troubleshooting
Achieving the perfect H&E stain is an art. As outlined by experts at Leica Biosystems, quality control is essential at every stage, even before staining.
Fixation is Key: The H&E procedure's success is highly dependent on proper specimen fixation. Poor or delayed fixation will lead to nuclear bubbling and pale staining.
Reagent Quality: Regularly monitor and change your reagents. Xylene and ethanol baths will become contaminated with water and wax, leading to poor deparaffinization and hazy slides. Hematoxylin can oxidize, losing its staining power.
Water Quality: Use high-quality, pure water (distilled or deionized) for reagent preparation and final rinses.
Troubleshooting: Pale Nuclei: This can be caused by old/exhausted hematoxylin, insufficient staining time, or over-differentiation in the acid-alcohol step.
Troubleshooting: Pale Cytoplasm: This is often due to rinsing for too long after the eosin step.
Troubleshooting: Hazy/Milky Slides: This is a classic sign of incomplete dehydration. If any water remains in the tissue when it enters the xylene, the slide will appear milky. Go back through the dehydration steps (100% ethanol) to correct it.