I. Purpose and Scope
This SOP is intended for routine hematoxylin–eosin (H&E) staining of paraffin sections and frozen sections. Under standard conditions, H&E staining uses basic hematoxylin to selectively visualize cell nuclei and other basophilic components, and acidic eosin to stain cytoplasm and extracellular matrix and other eosinophilic components. It is the most fundamental and commonly used staining method in histology, embryology, and pathology teaching and research.
II. Principle
Under conventional staining conditions (pH approximately 6), negatively charged acidic components within cells (e.g., DNA, RNA, cartilage matrix) exhibit affinity for basic dyes and are therefore basophilic; positively charged sites in proteins (e.g., hemoglobin, collagen fibers, muscle fibers, eosinophilic granules) exhibit affinity for acidic dyes and are therefore eosinophilic.
After forming a metal–dye complex (e.g., with alum) and undergoing oxidation, hematoxylin becomes a basic dye that binds to phosphate groups of DNA and nucleic acids, staining nuclei and certain calcium salts and mucinous components blue to blue-purple. Eosin Y is an acidic dye that ionizes in water to form negatively charged anions, which bind to cationic sites of proteins, staining cytoplasm, erythrocytes, muscle fibers, collagen fibers, etc. in varying intensities of red or pink. By a two-step staining sequence (nucleus first, cytoplasm second), together with appropriate differentiation and bluing, tissue architecture and pathological changes can be clearly demonstrated.
III. Reagents and Instruments
3.1 Main Reagents
(1) Eosin alcoholic staining solution (0.5%–1%)
Weigh 0.5–1 g Eosin Y, dissolve in a small amount of distilled water, then slowly add glacial acetic acid dropwise until a paste forms. Filter through filter paper, dry the residue in an oven, then bring to 100 mL with 95% ethanol (or absolute ethanol). Mix thoroughly until fully dissolved; set aside for use.
(2) Hematoxylin staining solution (example formulation, approximately 3000 mL)
Hematoxylin 6 g; absolute ethanol 100 mL; aluminum potassium sulfate 150 g; distilled water 2000 mL; sodium iodate 1.2 g; glacial acetic acid 120 mL; glycerol 900 mL.
Preparation: First dissolve hematoxylin in absolute ethanol. Dissolve aluminum potassium sulfate in distilled water and then mix with glycerol. Add the hematoxylin–ethanol solution, and finally add glacial acetic acid and sodium iodate. Mix thoroughly, allow to ripen (mature), then filter before use.
(3) 1% acid alcohol differentiator
Add 1 mL concentrated hydrochloric acid to 99 mL 70% ethanol, mix well, and set aside.
(4) Bluing solution / “return blue” solution
A weakly alkaline solution, such as running tap water, an ammonia water–tap water mixture, or an alkaline buffer; the specific formulation is determined according to laboratory routine practice.
(5) Xylene (or equivalent clearing agent), absolute ethanol, 95% ethanol, 80% ethanol, distilled water.
(6) Neutral mounting medium (neutral balsam) or other suitable mounting media.
3.2 Main Instruments
Routine tissue processing and sectioning equipment (paraffin embedding station, rotary microtome, or cryostat), incubator or drying oven, microscope, fume hood, timer, and organic-solvent-resistant staining racks/coplanar staining carriers.
IV. Procedure
4.1 H&E Staining of Paraffin Sections
4.1.1 Deparaffinization to Water
Place slides sequentially into each solution with gentle agitation:
(1) Xylene (I) 15 min.
(2) Xylene (II) 15 min.
(3) Xylene:absolute ethanol = 1:1 mixture 2 min.
(4) 100% ethanol (I) 5 min.
(5) 100% ethanol (II) 5 min.
(6) 80% ethanol 5 min.
(7) Distilled water 5 min, fully rehydrate.
4.1.2 Staining and Differentiation
(1) Stain in hematoxylin solution for 5 min.
(2) Rinse thoroughly in running tap water or distilled water for 5–10 min (at least 5 min under running water) to remove free dye and initiate partial bluing.
(3) Differentiate in 1% acid alcohol for 30 s with gentle agitation; observe nuclear staining becoming lighter to avoid over-differentiation.
(4) Rinse in water for 30 s to stop differentiation.
(5) Quickly dip in distilled water for approximately 5 s to remove residual acidic components.
(6) Stain in 0.5% eosin alcoholic solution for 1–3 min; adjust time according to the desired cytoplasmic intensity.
(7) Briefly rinse in distilled water for 30 s to remove excess eosin and prevent excessive background staining.
4.1.3 Dehydration, Clearing, and Mounting
(1) Brief rinse in 80% ethanol for 30 s.
(2) 95% ethanol (I) 1 min.
(3) 95% ethanol (II) 1 min.
(4) Absolute ethanol (I) 3 min.
(5) Absolute ethanol (II) 3 min, to ensure complete dehydration.
(6) Xylene (I) 3 min.
(7) Xylene (II) 3 min, until sections are fully cleared.
(8) Add neutral mounting medium to mount; gently place the coverslip, avoid air bubbles, and allow to dry at room temperature.
4.2 H&E Staining of Frozen Sections
4.2.1 Fixation and H&E Staining
(1) Fix frozen sections in an appropriate fixative for 10–30 s (e.g., cold acetone or an alcohol-based fixative, selected according to laboratory routine practice).
(2) Briefly rinse with distilled water or tap water for 1–2 s to remove excess fixative.
(3) Stain in hematoxylin solution (pre-warmed to 60℃) for 30–60 s.
(4) Quickly rinse off excess hematoxylin under running water for 5–10 s.
(5) Differentiate in 1% acid alcohol for 1–3 s, rapidly controlling according to nuclear intensity.
(6) Briefly rinse in water for 1–2 s.
(7) Blue in bluing solution for 5–10 s until nuclei appear clearly blue.
(8) Rinse under running water for 15–30 s.
(9) Stain in 0.5% phloxine (or eosin) staining solution for 30–60 s.
(10) Briefly rinse with distilled water for 1–2 s.
4.2.2 Dehydration, Clearing, and Mounting
(1) 80% ethanol 1–2 s.
(2) 95% ethanol 1–2 s.
(3) Absolute ethanol 1–2 s for rapid dehydration.
(4) Carbol-Xylene (phenol–Xylene) 2–3 s (if not used, absolute ethanol may be used instead, but ensure subsequent Xylene steps fully replace ethanol).
(5) Xylene (I) 2–3 s.
(6) Xylene (II) 2–3 s.
(7) Mount with neutral mounting medium and allow to dry at room temperature for use.
V. Staining Results and Interpretation
5.1 Normal Staining Characteristics
Nuclei: bright blue to deep blue with clear nuclear detail; cartilage matrix, areas of calcium salt deposition, and mucinous components are often dark blue or gray-blue. Cytoplasm: most cytoplasm appears pink to rose-red; eosinophilic granules are bright vivid red. Collagen fibers: pale pink. Elastic fibers: bright pink. Erythrocytes: orange-red. Proteinaceous exudate: pink. The degree of eosinophilia and basophilia may vary among tissues and disease states; for example, immature cells may show paler eosin staining or mild basophilia in the cytoplasm, whereas senescent or degeneratively changed cells often exhibit increased eosinophilia.
5.2 H&E Staining Evaluation Criteria
(1) The section is intact, with a thickness of 4–6 μm, uniform thickness, and no obvious wrinkles, knife marks, or cracks.
(2) Clear nuclear–cytoplasmic contrast: nuclei are sharp with appropriate blue intensity; the red–blue contrast of cytoplasm and matrix is balanced, neither purplish nor excessively red.
(3) Adequate dehydration and clearing: after mounting, the background is transparent and clear, without whitish haze or water-cloudiness; no obvious air bubbles or debris are present in the field.
(4) Secure mounting: mounting medium is evenly distributed, with no large-scale cracking or edge detachment.
VI. Precautions and Common Problems
(1) Deparaffinization and dehydration must be complete; otherwise, a whitish haze and blurred fields may appear after mounting and long-term preservation may be compromised. If necessary, appropriately extend the time in absolute ethanol and Xylene.
(2) Differentiation time in acid alcohol must be strictly controlled. If nuclear staining is too dark, a brief additional differentiation can be performed; if over-differentiated, nuclei become pale or even negative and restaining is required. It is recommended to use control slides initially to determine the optimal timing.
(3) Bluing should be completed under weakly alkaline conditions. Differences in tap water quality may affect results; if needed, use a standardized bluing solution formulation.
(4) Overstaining with eosin causes an excessively dark background and obscures tissue detail; understaining yields faint cytoplasmic coloration. Fine-tune the time according to specimen type.
(5) Carbol-Xylene has strong dehydrating capability, but exposure time should not be prolonged to avoid fading or tissue brittleness; it should be thoroughly removed by multiple Xylene changes thereafter.
(6) All operations involving organic solvents should be performed in a fume hood with appropriate fire safety measures and personal protective equipment.
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