Hematoxylin-Eosin (HE) Staining Kit (with Differentiating Solution)

Cat. No.: H1508460

GRADE & PURITY BioReagent, Biological Stain, for Microscopy

Overview

  Combined staining with Hematoxylin and Eosin, referred to as HE staining for short, is the most basic staining method in routine pathological slide preparation and has extremely wide applications. Hematoxylin is a tawny crystal extracted from logwood, a plant native to Central and South America. It is a basic dye that forms hematein after oxidation. When used together with a mordant (commonly trivalent aluminum or iron salts), it can stain cell nuclei. In pathological diagnosis, teaching, and scientific research, HE staining is commonly used to observe the morphological structure of normal and diseased tissues. It can identify or differentiate certain abnormal substances and special components present in diseased tissues and cells. Special staining methods, enzyme histochemistry, immunohistochemistry, and other techniques are all performed on the basis of observing H&E-stained tissue sections. In H&E-stained sections, cell nuclei stain blue, and cytoplasm stains red, forming a distinct contrast that facilitates observation and analysis.

  The Hematoxylin-Eosin (HE) Staining Kit (with Differentiating Solution) contains hematoxylin staining solution formulated with an independently developed recipe. The solution is composed of imported high-purity hematoxylin and oxidants, and is free of harmful substances such as mercuric oxide and methanol. It exhibits excellent staining effects on cell nuclei with the characteristics of being less prone to precipitation and metallic film formation. It has a broad application range, suitable for use in human, animal, livestock, and aquatic research fields, and can be applied to the staining of tissue paraffin sections, frozen sections, and tissue cells. Both hematoxylin staining solution and eosin staining solution are reusable.Note: This product is for research use only and not intended for clinical diagnosis or other purposes.

Staining Principles

1. Nuclear Staining Principle: Hematoxylin is a naturally occurring basic dye that can stain cell nuclei. The main component of chromatin in the nucleus is DNA. In the DNA double helix structure, the phosphate groups on the two nucleotide chains face outward, making the outer surface of the DNA double helix negatively charged (acidic). This allows it to easily bind to positively charged basic hematoxylin dye via ionic or hydrogen bonds, resulting in staining. Hematoxylin appears blue in alkaline solutions, so cell nuclei are stained blue.

2. Cytoplasmic Staining Principle: Eosin is a synthetic acidic dye that can stain cytoplasm under certain conditions. The main component of cytoplasm is protein, an amphoteric compound. Cytoplasmic staining is closely related to the pH of the staining solution. When the pH of the staining solution is below the isoelectric point of cytoplasmic proteins (4.7-5.0), cytoplasmic proteins undergo basic ionization and carry a positive charge, enabling them to be stained by negatively charged acidic dyes. Eosin dissociates into negatively charged anions in water, which bind to positively charged cations of cytoplasmic proteins, staining the cytoplasm red.

3. Differentiation Principle: After staining, the process of removing excess bound dye from tissues using specific solutions is called differentiation, and the solution used is called a differentiating solution. In HE staining, 0.5-1% hydrochloric acid alcohol is commonly used as the differentiating solution. Acids can destroy the quinone structure of hematoxylin, causing the dye to dissociate from tissues and fade. Most tissues must be differentiated with hydrochloric acid alcohol after hematoxylin staining to remove excess hematoxylin bound to cell nuclei and hematoxylin adsorbed by cytoplasm before eosin staining. This step ensures clear differentiation between nuclear and cytoplasmic staining.

4. Bluing Principle: After differentiation, hematoxylin exists in a red ionic state (appearing red) under acidic conditions and in a blue ionic state (appearing blue) under alkaline conditions. Tissue sections turn red or pink after differentiation with acidic alcohol. Immediately rinse the sections with water to remove acid and terminate differentiation, then treat them with weakly alkaline water to restore the blue color of hematoxylin-stained nuclei. This process is called bluing or regeneration of blue color. Alternatively, rinsing with tap water (especially warm water) can also achieve bluing, but it requires a longer time.

Materials to Be Prepared by User

1. Xylene or paraffin-embedded deparaffinization and clearing solution, graded ethanol, bluing solution, tap water or distilled water

2. Ether-ethanol mixed fixative, 4% paraformaldehyde, neutral balsam or environment-friendly mounting medium

Operating Procedures (For Reference Only)

(1) Staining of Paraffin Sections

1. Deparaffinization and Hydration of Sections

① Treat sections with xylene or paraffin-embedded deparaffinization and clearing solution twice, 5-10 minutes each time.

② (Optional) Treat with anhydrous ethanol twice, 3-5 minutes each time.

③ 95% ethanol: 3-5 minutes.

④ 90% ethanol: 3-5 minutes.

⑤ 80% ethanol: 3-5 minutes.

⑥ Rinse with tap water or distilled water (30-40℃ warm water is also acceptable): 1-3 minutes.

2. Staining

① Stain with hematoxylin staining solution: 3-8 minutes.

② Rinse with tap water or distilled water: 5-10 seconds.

③ Differentiate with acid alcohol: 2-5 seconds.

④ Rinse with tap water: 20-30 seconds.

⑤ Bluing with bluing solution or warm water: 20-40 seconds.

⑥ Rinse with tap water: 30-60 seconds.

⑦ Stain with aqueous eosin staining solution: 20-60 seconds.

⑧ Rinse with tap water: 30-60 seconds.

3. Dehydration, Clearing and Mounting

① 80% ethanol: 10-20 seconds.

② 90% ethanol: 10-20 seconds.

③ 95% ethanol twice, 1-2 minutes each time.

④ Anhydrous ethanol twice, 2-3 minutes each time.

⑤ Clear with xylene or paraffin-embedded deparaffinization and clearing solution three times, 2-3 minutes each time.

⑥ Mount with neutral balsam.

(2) Staining of Frozen Sections

1. Fix with ether-ethanol mixed fixative: 5-10 seconds.

2. Rinse with tap water: 2-5 seconds.

3. Stain with hematoxylin staining solution (dropwise application) for 1-5 minutes (heating to 50℃ is optional).

4. Rinse with tap water: 2-5 seconds.

5. Differentiate with acid alcohol: 2-5 seconds.

6. Rinse with tap water: 2-5 seconds.

7. Bluing with bluing solution or warm water: 2-5 seconds.

8. Rinse with tap water: 5-10 seconds.

9. Stain with aqueous eosin staining solution: 2-20 seconds.

10. Rinse with tap water: 5-10 seconds.

11. 80% ethanol: 1-2 seconds.

12. 95% ethanol: 1-2 seconds.

13. Anhydrous ethanol: 2-5 seconds.

14. Phenol-xylene (1:3): 2-5 seconds.

15. Clear with xylene or paraffin-embedded deparaffinization and clearing solution three times, 2-5 seconds each time.

16. Mount with neutral balsam.

(3) Cell Staining

1. Fix with 4% paraformaldehyde for 10-20 minutes.

2. Rinse with tap water twice, 2 minutes each time.

3. Rinse with distilled water twice, 2 minutes each time.

4. Follow the staining, deparaffinization, clearing, and mounting steps for paraffin sections, with appropriately shortened incubation times.

Staining Results

Cell Nuclei: Blue

Cytoplasm, Muscle Fibers, Collagen Fibers, Thyroid Colloid, etc.: Red of varying intensities

Keratin, Red Blood Cells, etc.: Bright Orange-Red

Precautions

1. Ensure thorough deparaffinization of sections. At low temperatures, sections can be treated in an incubator at 60-70℃.

2. Replace graded ethanol with fresh solutions regularly.

3. The duration of acid alcohol differentiation should be adjusted according to section thickness, tissue type, and reagent freshness. In addition, the tap water rinsing time after differentiation should be sufficient to completely remove residual acid.

4. The ether-ethanol mixed fixative is prepared by mixing equal volumes of ether and 95% ethanol, adding an appropriate amount of acetic acid, and storing it in a sealed container.

5. Minimize staining time for frozen sections.

6. Bluing solution can be replaced with 0.2-1% ammonia water, Scott’s bluing solution, or 0.1-1% lithium carbonate solution.

7. For your safety and health, wear a lab coat and disposable gloves during operation.

Specifications

Synonyms

Hematoxylin-Eosin (HE) Staining Kit

Specifications & Purity

BioReagent,for microscopy,Biological Stain

Grade

Biological Stain, BioReagent, for Microscopy

Application

Cell Stain

Storage and Shipping

Storage

Protected from light,Room temperature

Shipped In

Normal

Stability And Storage

Each component has a shelf life of 1 year under corresponding storage conditions.

Contents & Storage

H1508460	Component	3×100 mL	3×500 mL	Storage
H1508460A	Hematoxylin Staining Solution	100 mL	500 mL	RT. Store in the dark.
H1508460B	Acid Alcohol Differentiating Solution	100 mL	500 mL	RT
H1508460C	Aqueous Eosin Staining Solution	100 mL	500 mL	RT. Store in the dark.

Application Protocols

Not specified for this item; refer to CoA/Spec Sheet.

Note: As a general remark (not a product protocol), H&E workflows typically include deparaffinization, rehydration through graded alcohols to water, hematoxylin staining, differentiation (regressive methods), bluing, eosin counterstaining, dehydration, clearing, and mounting. Follow the manufacturer’s instructions supplied with your lot and validate timings on in-house control tissues.

Biological Roles

The kit’s components are synthetic/natural dyes used to differentiate tissue structures; they do not serve as biological cofactors or metabolites in the staining context.

Literature-based staining biology (not product specifications):

• Hematoxylin (oxidized to hematein) complexes with metal mordants (e.g., Al3+) to form cationic dye lakes that bind to polyanionic sites in nuclei (phosphate backbone of DNA/RNA), yielding blue–purple basophilic staining following an alkaline “bluing” step.
• Eosin Y is an anionic xanthene dye that binds to basic proteins and cytoplasmic components, producing pink to red eosinophilic staining. Collagen, muscle, and erythrocytes display characteristic shades that assist in structural delineation under brightfield microscopy.
• The differentiating solution selectively removes excess nuclear dye from non-nuclear regions in regressive protocols, enhancing nuclear–cytoplasmic contrast.

Applications in research biology:

• Morphometry, tissue architecture assessment, and as a baseline counterstain prior to advanced assays (e.g., special stains, IHC/ISH in research settings). The balanced coloration aids in recognizing cellular compartments and extracellular matrices.

Note: No medical or diagnostic claims are made. This product is designated For research use only (Product Data). Component-specific biochemical interactions and any proprietary additives are formulation-dependent; consult the CoA/Spec Sheet for details.

Buffer Applications

This kit is not a general-purpose buffering system. However, H&E workflows typically include a brief alkaline “bluing” step after hematoxylin and before eosin to convert reddish-purple hemalum to a crisp blue (literature, general):

• Common bluing solutions (not supplied information; verify your workflow):
  • Mild alkaline tap water substitutes (e.g., Scott’s tap water, lithium carbonate water, or dilute ammonia water) in the pH ~8–9 range.
• Practical guidance (literature):
  • Overly strong alkalinity can cause background haze or partial destaining; adjust exposure empirically for your sections.
  • Maintain separate containers for hematoxylin rinse and bluing to avoid cross-contamination; refresh solutions at defined intervals.

For this specific kit, any included buffers or recommended bluing reagents are not specified; refer to the CoA/Spec Sheet and your laboratory SOP.

Green Alternatives

While the H&E dyes themselves are established, environmental impact in H&E workflows is often dominated by solvents used for dehydration and clearing. The following points reflect general, literature-based sustainability options (not product specifications):

• Reduce aromatic hydrocarbons:
  • Traditional clearing with xylene can be replaced by validated alternatives such as aliphatic hydrocarbon clears or d-limonene-based substitutes. Trade-offs include odor, drying time, and compatibility with mounting media.
• Optimize alcohol usage:
  • Consider isopropanol in place of ethanol where validated, or reduce the number/volume of graded alcohol baths through process optimization and reuse policies consistent with your QA and local regulations.
• Aqueous workflows:
  • Use aqueous eosin formulations and water-based mounting media when optical and archival requirements permit. This may eliminate the final xylene step but can affect long-term slide stability and refractive index matching.
• Waste minimization:
  • Implement closed containers, rotation schemes for differentiator baths, and on-site solvent recycling where permitted. Segregate dye-containing aqueous waste from solvent waste to facilitate treatment.

Comparison (literature, general):

• Xylene: excellent clearing and rapid drying; higher VOC/worker exposure concerns; hazardous waste stream.
• Aliphatic substitutes: lower aromatic content and odor; may require longer clearing times and careful matching to mounting medium.
• Aqueous mountants: reduced VOCs; potentially less archival robustness and different optical characteristics.

Validate any change with pilot runs and QC slides to preserve staining quality.

Pharmaceutical Uses

Not typically applicable. H&E staining kits are laboratory reagents for tissue staining and visualization under light microscopy and are not used as pharmaceutical excipients or therapeutics.

Research/manufacturing context (literature, general):

• In pharmaceutical R&D and biologics manufacturing support, H&E staining may be employed in nonclinical research and in-process investigations to examine tissue morphology or assess sample integrity. Such use is procedural and not part of a drug product formulation.

Regulatory note:

• This product is designated For research use only (Product Data). Any use in regulated workflows requires end-user validation and appropriate quality documentation.

No pharmacopeial monograph applicability is provided for this kit; consult your QA unit if a validated, compliant staining SOP is required.

Physical Properties

Item-specific physico-chemical specifications are not provided for this kit.

• Appearance: Not specified for this item; refer to CoA/Spec Sheet.
• Density, refractive index, pH, solvent composition: Not specified for this item; refer to CoA/Spec Sheet.
• Boiling/flash points: Not specified for this item; refer to SDS for each component.

General literature information (for context only; formulation-dependent and not product specifications):

• Hematoxylin/hematein solutions are typically aqueous or hydroalcoholic and may be formulated with an aluminum mordant; pH of working solutions often ranges from ~2.5–3.5 (progressive) to slightly higher with certain “regressive” formulations (literature).
• Eosin Y working solutions are commonly prepared in aqueous or alcoholic media (ethanol or isopropanol) with pH ~4.0–5.5 to optimize staining contrast (literature).
• Differentiating solutions in H&E workflows are typically dilute acidified alcohols (e.g., 0.1–1% HCl in 70–95% alcohol) used briefly to remove excess nuclear stain (literature). Composition and strength vary by supplier and application.

Solubility and miscibility (literature, general):

• Hematoxylin/hematein formulations are fully miscible with water; hydroalcoholic variants mix with common lower alcohols.
• Eosin Y solutions are typically miscible with water and lower alcohols.

Note: For authoritative, component-specific values and any acceptance criteria, consult the item’s CoA/Spec Sheet and SDS.

Quality & Grades

• Stated grade (Product Data): BioReagent, Biological Stain, for Microscopy.
• What this implies:
  • BioReagent/biological stain grade indicates suitability for laboratory staining of biological specimens, with attention to color strength, low background, and consistency between lots.
  • “For Microscopy” denotes that the reagents are intended for histological/cytological staining workflows (light microscopy) and are not for clinical diagnostic use unless specifically validated by the end user.
• Acceptance criteria and composition: Not specified for this item; refer to CoA/Spec Sheet for component concentrations, dye content, and any lot-release quality attributes (e.g., performance on standard tissue controls).
• Stabilizers/mordants: Not specified for this item; refer to CoA/Spec Sheet. Many hematoxylin formulations include aluminum salts and proprietary stabilizers to control oxidation state and shelf-life (literature, general remark).
• Practical guidance:
  • Verify lot-to-lot performance on your in-house control sections (e.g., spleen, liver, kidney) to confirm nuclear crispness and cytoplasmic contrast.
  • Record exposure times optimized for your fixation/processing pipeline; alcohol percentage in eosin and the strength of differentiator significantly affect contrast and background.
• Documentation: For audits and reproducibility, archive the item’s CoA, SDS, and any internal QC images. This kit is intended For research use only (Product Data).

Reaction & Applications

This product is not a chemical reagent for synthesis; it is a specialized staining kit for histology and cytology.

Research/lab applications (literature, general):

• Routine H&E staining of paraffin-embedded or frozen tissue sections to visualize tissue architecture.
  • Hematoxylin/hemalum provides basophilic nuclear staining (chromatin, nucleoli) with crisp, blue–purple coloration after “bluing.”
  • Eosin provides acidophilic counterstaining of cytoplasm, extracellular matrix (collagen), and erythrocytes, yielding pink to red hues.
• Progressive vs regressive methods:
  • Progressive: sections are stained to end-point with hematoxylin then blued; minimal or no differentiation.
  • Regressive: sections are overstained with hematoxylin, then precisely differentiated with an acidic alcohol to sharpen nuclear contrast (this kit includes a differentiating solution to support regressive workflows).
• Use cases:
  • Morphological assessment in basic research, toxicologic pathology research, and method development for additional special stains or IHC (sequence H&E to assess tissue quality prior to downstream labeling; research use only).
• Practical considerations (general):
  • Section thickness (e.g., 3–6 µm for paraffin), fixation chemistry (10% NBF vs alternatives), and processing (dehydration/clearing) strongly influence color balance and background.
  • Control differentiation time by tissue type; delicate epithelia require shorter differentiation than dense, highly basophilic tissues.

Note: Manufacturer Applications were not specified in Product Data; adapt these literature practices to your lab SOPs and consult the kit’s CoA/Spec Sheet for product-specific guidance.

Reaction Conditions

Not applicable. This product is not used for chemical reactions. It is supplied for histological staining procedures.

Operational variables relevant to H&E staining (literature, general; not product-specific):

• Section type/thickness: paraffin sections typically 3–6 µm; frozen sections thinner.
• Timing: progressive vs regressive exposure times vary by fixation, tissue type, and dye strength. Differentiation is usually brief (seconds) followed by immediate rinsing and bluing.
• Temperature: ambient.
• Atmosphere: not critical; avoid excessive evaporation of alcohol-containing solutions.

For kit-specific timings, temperatures, and workflow, consult the CoA/Spec Sheet or vendor protocol.

Safety & Handling

• GHS classification, signal word, H/P statements, and pictograms: Not specified for this item; refer to the SDS for each kit component.
• Storage conditions (Product Data): Protected from light; room temperature. Ship: Normal.
• General hazards (literature/good practice; not product-specific):
  • Hematoxylin/eosin dyes can cause skin and eye irritation; avoid inhalation or ingestion. Some formulations may contain alcohols and/or acids (differentiator), which can be flammable and/or corrosive depending on concentration.
  • Differentiating solutions often contain dilute mineral acid in alcohol; treat as potentially flammable and corrosive. Avoid contact with bases, strong oxidizers, and ignition sources. Use spark-free equipment around alcohol-containing reagents.
• PPE: Wear lab coat, safety glasses or face shield, and appropriate chemically resistant gloves (e.g., nitrile). Work in a well-ventilated area or fume hood when handling volatile or acidic components.
• Light sensitivity: Keep bottles tightly closed and protected from light to preserve dye integrity and color balance.
• Spill/first aid (general):
  • Skin/eye contact: Rinse with copious water for at least 15 minutes; remove contaminated clothing; seek medical attention as needed.
  • Inhalation: Move to fresh air; obtain medical advice if symptoms persist.
  • Ingestion: Rinse mouth; do not induce vomiting; seek medical attention.
• Waste: Collect dye- and alcohol-containing wastes in compatible, labeled containers for solvent/acid waste disposal per institutional and regulatory requirements.

Always defer to the component SDS and local EHS guidance for authoritative safety instructions.

Solvent Selection

As a ready-to-use staining kit, solvent selection is largely dictated by the provided formulations and your tissue processing workflow rather than by reaction solvent choice.

General, literature-based compatibility notes (not product specifications):

• Polarity/miscibility:
  • Hematoxylin solutions are typically aqueous or hydroalcoholic and fully miscible with water and lower alcohols.
  • Eosin solutions are commonly aqueous or alcoholic (ethanol/isopropanol), miscible with water and alcohols. Alcohol-rich eosin often yields faster penetration and crisper cytoplasmic staining.
  • Differentiating solutions are frequently acidified alcohols; miscible with water and alcohol. Rinse thoroughly to stop differentiation.
• Clearing and dehydration (workflow context):
  • Pre- and post-stain solvent steps typically involve graded alcohols (70–100% ethanol or isopropanol) for dehydration and xylene or xylene substitutes for clearing (literature). Choice affects mounting compatibility and long-term slide stability.
• When to choose which medium (literature):
  • Aqueous eosin can give slightly warmer tones and may reduce flammability risk; alcoholic eosin provides rapid staining and consistent differentiation when working with high-throughput tissue processors.
  • If minimizing volatile aromatic exposure, consider aliphatic or citrus-based clearing agents validated in your lab; verify optical matching with the mounting medium.

For this item’s exact solvent composition and any restrictions, consult the CoA/Spec Sheet and SDS.

Storage & Reconstitution

• Storage conditions (Product Data): Protected from light; room temperature.
• Shipping (Product Data): Normal.
• Reconstitution: Not specified for this item; refer to CoA/Spec Sheet. The kit typically contains ready-to-use liquid reagents; if concentrates are provided, follow the lot-specific dilution instructions on the label/CoA.
• Handling:
  • Keep containers tightly closed to minimize evaporation and contamination, especially for alcohol-containing or acidic components.
  • Protect from light to preserve dye stability and color balance.
• Stability/shelf-life: Not specified for this item; refer to CoA/Spec Sheet.
• Freeze–thaw: Not applicable for room-temperature liquid reagents unless otherwise stated; avoid freezing unless the CoA explicitly permits it.
• Before use: Mix gently to ensure homogeneity; avoid introducing bubbles. Inspect for precipitates or color changes; if observed, consult SDS/CoA and your QC policy before use.

For definitive instructions on storage duration after opening, dilutions (if any), and disposal of expired solutions, consult the item’s CoA/Spec Sheet and follow institutional SOPs.

Structure & Identity

This listing is a multicomponent Hematoxylin–Eosin (H&E) staining kit that includes a differentiating solution. As a kit, it does not correspond to a single molecular structure.

• Item type: Multicomponent histology staining kit (hematoxylin stain, eosin counterstain, plus a differentiating solution)
• Molecular formula: Not applicable to a kit; component-level formulas are mixture-dependent (Not specified for this item; refer to CoA/Spec Sheet.)
• Molecular weight: Not applicable for a kit (Not specified for this item; refer to CoA/Spec Sheet.)
• SMILES / InChI / InChIKey: Not applicable for a kit containing multiple formulated solutions.
• Structural features (general, literature):
  • Hematoxylin is a natural dihydroxyanthraquinone-type phenolic compound that, upon oxidation to hematein and complexation with a mordant (e.g., Al3+), forms a cationic “lake” that binds basophilic substrates (DNA/RNA–rich structures).
  • Eosin Y is a xanthene dye (tetrachlorinated fluorescein derivative) with anionic character that binds to basic (eosinophilic) proteins.
• Formulation/2D description: The kit provides pre-formulated liquid reagents; there is no single 2D structure to depict. Component identities and concentrations are formulation-specific (Not specified for this item; refer to CoA/Spec Sheet.).
• CAS / CID: Not specified for this item; refer to CoA/Spec Sheet.

Synthetic Utility

Not applicable. This product is a histological staining kit and is not intended as a reagent for chemical synthesis or building-block transformations.

If you require hematoxylin or eosin as discrete chemicals for synthesis or analytical method development, source those individual materials with appropriate specifications and SDS. This kit’s formulations are optimized for staining performance rather than synthetic utility.

Target Specificity

Not applicable. This product is not an antibody or affinity reagent and has no antigen/epitope specificity. H&E is a general morphological stain that differentially colors basophilic and eosinophilic tissue components (literature, general). No clone, isotype, or species reactivity applies.