Hematoxylin and Eosin combined staining, referred to as HE staining, is the most fundamental staining method in routine pathological section preparation and has extremely wide applications. Hematoxylin is a pale yellowish-brown crystalline substance extracted from logwood native to Central and South America. As a basic dye, it is oxidized to form hematein, which, when used together with a mordant (commonly trivalent aluminum or iron salts), 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 enables the identification or differentiation of certain abnormal substances and special components present in diseased tissues and cells. Moreover, special staining methods, enzyme histochemistry, immunohistochemistry, and other techniques are all performed on the basis of observing HE-stained tissue sections. In HE-stained sections, cell nuclei appear blue and cytoplasm appears red, forming a sharp contrast that facilitates observation and analysis.
Hematoxylin-Eosin (HE) High-Definition Consistent Staining Kit (High-Definition Consistent Staining) consists of pre-stain solution, hematoxylin staining solution, differentiating solution Ⅰ, differentiating solution Ⅱ, bluing solution, and eosin staining solution. Among them, the hematoxylin staining solution adopts a proprietary formula independently developed by Leigen, composed of imported high-purity hematoxylin and oxidants, without harmful substances such as mercuric oxide and methanol, and exhibits excellent staining effects on cell nuclei. This product is suitable for displaying tissue morphological structures in routine HE section staining. It features firm dye binding, non-fading after long-term storage, and consistent staining results for 1 to 2500 sections, which is conducive to the standardization and unification of HE staining. 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 stains 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). It readily binds to positively charged basic hematoxylin dye via ionic bonds or hydrogen bonds, thus being stained. Hematoxylin appears blue in alkaline solutions, so cell nuclei are stained blue.
2. Cytoplasmic Staining Principle: Eosin is a synthetic acidic dye that stains cytoplasm under certain conditions. Cytoplasm is mainly composed of proteins, which are amphoteric compounds. Cytoplasmic staining is closely related to the pH value 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, allowing 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 Effect: 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. The acid destroys the quinone structure of hematoxylin, causing the dye to dissociate from the tissue and fade. Most tissues must be differentiated with hydrochloric acid alcohol after hematoxylin staining to remove excess hematoxylin bound to nuclei and hematoxylin adsorbed to cytoplasm before eosin staining, ensuring clear distinction between nuclear and cytoplasmic staining.
4. Bluing Effect: After differentiation, hematoxylin exists in a red ionic state (red color) under acidic conditions and in a blue ionic state (blue color) under alkaline conditions. Tissue sections appear red or pink after differentiation with acid alcohol. The differentiation is immediately terminated by rinsing the sections with water to remove acid, followed by treatment with weakly alkaline water to restore the blue color of hematoxylin-stained nuclei. This process is called bluing or alkalization. Additionally, rinsing with tap water (especially warm water) can also achieve bluing, but it requires a longer time.
H1508526
| Component | 6×100 mL | 6×500 mL | Storage |
| H1508526A | Pre-stain Solution
| 100 mL | 500 mL | RT. Store in the dark.
|
| H1508526B | Hematoxylin Staining Solution
| 100 mL | 500 mL | RT. Store in the dark. |
| H1508526C | Differentiating Solution Ⅰ
| 100 mL | 500 mL | RT. |
| H1508526D | Differentiating Solution Ⅱ
| 100 mL | 500 mL | RT. |
| H1508526E | Bluing Solution
| 100 mL | 500 mL | RT. |
| H1508526F | Eosin Staining Solution
| 100 mL | 500 mL | RT. Store in the dark. |
Materials to Be Prepared by the User
1. Ether-ethanol mixed fixative, 4% paraformaldehyde, tap water or distilled water
2. Environmentally friendly dewaxing solution, environmentally friendly clearing solution, graded ethanol solutions, neutral gum or environmentally friendly mounting medium
Operating Procedures (For Reference Only)
(1) Paraffin Section Staining
1. Dewaxing and Hydration of Sections
① Dewax with environmentally friendly dewaxing solution twice, 5-10 minutes each time.
② Rinse with anhydrous ethanol twice, 1-3 minutes each time.
③ 95% ethanol: 1 minute.
④ 90% ethanol: 1 minute.
⑤ 80% ethanol: 1 minute.
⑥ Rinse with tap water or distilled water: 1 minute.
2. Staining
① Treatment with pre-stain solution: 30 seconds.
② Staining with hematoxylin staining solution: 5 minutes.
③ Rinse with tap water or distilled water: 2 minutes.
④ Differentiation with differentiating solution Ⅰ: 15 seconds.
⑤ Differentiation with differentiating solution Ⅱ: 5 seconds.
⑥ Rinse with tap water or distilled water: 2 minutes.
⑦ Bluing with bluing solution: 1 minute.
⑧ Rinse with tap water or distilled water: 1 minute.
⑨ 80% ethanol: 1 minute.
⑩ Staining with eosin staining solution: 10 seconds.
3. ehydration, Clearing and Mounting
① 95% ethanol: 1 minute.
② Rinse with anhydrous ethanol twice, 1 minute each time.
③ Clear with environmentally friendly clearing solution twice, 2 minutes each time.
④ Mount with neutral gum or environmentally friendly mounting medium.
(2) Frozen Section Staining
1. For frozen sections stored at -20℃, allow them to warm up at room temperature and air-dry slightly, then fix with methanol for 1 minute. For freshly prepared frozen sections, let them stand at room temperature for several minutes until the OCT embedding medium is completely air-dried before staining to prevent section detachment.
2. Rinse with tap water for 5 seconds.
3. Follow the pre-staining, staining, dehydration, clearing and mounting steps for paraffin sections, with appropriately shortened incubation times.
(3) Cell Staining
1. Fix cell slides with 4% paraformaldehyde for 5-10 minutes.
2. Rinse with tap water twice, 2 minutes each time.
3. Rinse with distilled water twice, 2 minutes each time.
4. Follow the pre-staining, staining, dehydration, clearing and mounting steps for paraffin sections, with appropriately shortened incubation times.
(4) Automated Staining
Follow the same operating procedures as paraffin section staining.
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 dewaxing of sections. When the temperature is low, treat sections in an incubator at 60-70℃.
2. Read the instruction manual carefully before use and use the product within its shelf life.
3. Replace graded ethanol solutions with fresh ones regularly.
4. Store the product at the specified temperature. If stored below 15℃, alum crystals may precipitate in the hematoxylin staining solution, which generally does not affect product quality but may reduce the number of sections that can be stained. If stored above 30℃, the oxidation rate of hematoxylin will accelerate, leading to decreased staining ability, and the volatilization of eosin staining solution will also increase.
5. The differentiation time with acid alcohol should be adjusted according to section thickness, tissue type, and reagent freshness. In addition, sufficient rinsing time with tap water after differentiation is required to completely remove residual acid.
6. It is recommended to use the product in conjunction with environmentally friendly dewaxing solution, environmentally friendly clearing solution and environmentally friendly mounting medium for experiments.
7. For your safety and health, wear a lab coat and disposable gloves during operation.
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