Hematoxylin staining solution

Product Description

Hematoxylin staining solution is formulated by combining several classic methods. This solution is mercury-free and can be used for immunohistochemical counterstaining as well as H&E staining.

Order Information

Transportation and Storage

Store at room temperature, protected from light. Shelf life: 12 months.

Instructions for Use

1. Reagent Preparation

Acid-alcohol solution: Add 1 mL of hydrochloric acid to 100 mL of 70% ethanol and store at room temperature. Note: After cryosectioning, ensure that all steps—drying, fixation in ethanol or methanol, heat fixation, and the use of charged or adhesive coverslips—prevent the tissue sections from detaching during staining.

2. 95% ethanol: Before beginning HE staining, tissue sections must be fixed and hydrated. Place the sections in 95% ethanol for approximately 2 minutes to initiate hydration and fixation of the tissue. Note: Omitting the use of 95% ethanol for hydration and fixation does not affect morphological details.

3. Hydration: Further deparaffinize the tissue sections for 3–5 minutes, then rinse off any residual ethanol from the previous step.

4. Hematoxylin: After hydration, tissue sections are stained with hematoxylin for 1–3 minutes; the staining time may be adjusted based on the staining results and experimental requirements. Hematoxylin is a basic dye that stains acidic cellular components—including nucleic acids, mucopolysaccharides, and acidic glycoproteins—producing a blue or bluish‑purple color. Note: Without hematoxylin, eosin would stain the tissue a bright pink, making the nuclei virtually invisible. Tissue architecture would be difficult to distinguish, and intracellular structures would largely disappear, rendering them indiscernible. To address these issues, a counterstain with hematoxylin can be applied.

(1) Weak staining is due to: inadequate self‑dissolution or poor fixation; excessive decalcification; insufficient staining time; over‑destaining (an excessively high acid‑to‑alcohol ratio or prolonged acid‑ethanol destaining); weak hematoxylin action (over‑oxidized or overly aged hematoxylin, or excessive dilution with water); contamination of the rinsing solution; sections that are too thin; or inadequate removal of ethanol or insufficient water rinsing prior to staining.

(2) Overstaining is caused by: drying of tissue sections; excessively high hematoxylin concentration; prolonged staining time; excessive mounting medium on the slide; insufficient or inadequate decolorization; overly thick sections; and prolonged exposure to heat.

5. Washing with water: After hematoxylin staining, rinsing the tissue with water is crucial. This step, along with subsequent washes, removes excess dye and mordants. Proper rinsing eliminates surface metallic sheen, which can inhibit hematoxylin staining. Note: Skipping this step may lead to undesirable outcomes, such as precipitate formation, dilution of the staining solution, and the presence of surface metallic sheen.

6. Acid-alcohol: Use acid-alcohol to differentiate for 3–5 seconds. In the hematoxylin staining procedure, if the tissue sections are intentionally over-stained, acid-alcohol will remove excess dye and background staining from the slide.

Note: If this step is omitted, the sections will appear dark purple, and acidophilic structures (such as collagen) will fail to stain a bright pink, making it difficult to distinguish between tissue components and compromising accurate interpretation of the slides. Additionally, over‑differentiation may result from excessively high acid concentration or prolonged decolorization; under‑differentiation, on the other hand, is due to insufficient acid concentration, inadequate decolorization time, or the presence of excess proteins or gelatin on the sections.

7. Washing: Wash with water for 30 seconds to terminate the acid-alcohol reaction and further prevent excessive removal of hematoxylin from the tissue sections. Note: If the sections are not rinsed, the acid-alcohol may cause over‑decolorization. Failure to remove the decolorizing agent will result in continued removal of hematoxylin from the tissue sections by the acid-alcohol.

8. Rinse with tap water or stain with blueing solution: Rinsing with tap water for 3–5 minutes can achieve blueing, converting the purplish‑red hematoxylin to a bluish‑purple color. Typically, blueing reagents are pH‑dependent and consist of alkaline solutions. Therefore, if tap water is used as the blueing agent, daily monitoring of its pH is essential, as tap water pH can fluctuate from day to day. Alternatively, use a blueing solution for 30 seconds to 1 minute, followed by running tap water rinsing for 5 minutes. Note: Skipping the blueing step will cause subtle changes in the hematoxylin stain’s color, making it difficult to distinguish. Instead of a deep blue, the cell nuclei may appear purple, and sometimes even red. Over‑blueing of the nuclei should be avoided. If tissues are excessively blue, this is usually due to an excess of hematoxylin in the tissue sections.

9. Washing: This step prepares for eosin counterstaining by rinsing with water for 30 seconds to remove excess bluing agent. In an alkaline environment, eosin cannot stain; removing the bluing agent allows the eosin to maintain its acidic pH, ensuring uniform counterstaining. Note: Without subsequent water washing after the bluing step, the tissue section will not stain properly with eosin. Consequently, acidic structures will appear pale and unevenly stained, leading to inaccurate interpretations.

10. Eosin: To accurately distinguish intracellular structures, eosin serves as an excellent counterstain to hematoxylin. Counterstain with eosin for 30–60 seconds; the staining time may be adjusted according to the staining results and experimental requirements. Eosin is an acidic dye that stains the cytoplasm, particularly mitochondria, secretory granules, and collagen. It can differentiate among various intracellular organelles and distinct types of connective tissue. Note: Failure to properly counterstain tissue sections with eosin will result in poorly differentiated sections.

(1) Weak staining is due to: excessively thin tissue sections; insufficient staining time; and excessive differentiation in 95% ethanol.

(2) Overstaining is caused by: a high dye concentration (possibly due to excessive evaporation); excessively thick tissue sections; prolonged staining time; and insufficient differentiation in 95% ethanol.

(3) Eosin staining of undifferentiated tissue (a single color) is due to: incomplete fixation; or over‑staining (prolonged staining time or excessively concentrated stain).

11. 95% ethanol: Treat with 95% ethanol for 2 seconds to 1 minute, adjusting the duration based on the staining results and experimental requirements; varying the differentiation time with eosin will produce different shades of pink. Note: If the 95% ethanol becomes diluted (e.g., due to carryover of eosin from the previous staining step), the differentiation effect will be suboptimal.

12. 100% Ethanol: Use 100% ethanol to decolorize the tissue sections. Treat with 100% ethanol for 1 minute, then rinse with fresh 100% ethanol for another 1 minute. Note: This step is often overlooked; without 95% ethanol, it can be difficult to distinguish acidic structures, and the tissue sections may turn pink. If 100% ethanol is omitted during dehydration, the sections will not be fully dehydrated, leading to the formation of water droplets beneath the coverslip. These droplets can combine with xylene in the next step to produce a white haze, which will compromise the quality of the tissue sections.

13. Xylene: Treat with xylene for 5 minutes. After thorough dehydration, xylene removes residual alcohol from tissue sections. Following alcohol removal, when mounting the slides, xylene can also serve as a clearing agent to ensure proper solubility. Due to its potential toxicity, alternative clearing agents such as limonene and cycloalkanes may be used. These alternatives provide comparable staining quality while being safer and easier to handle. Note: If this step is skipped and xylene clearing is omitted, alcohol will remain on the tissue sections, causing eosin to leach off the slides.

14. Mounting: Mount with neutral gum, allow to air-dry or dry in an oven, then examine under a microscope.

Staining results: The nucleus is stained blue; the cytoplasm is stained red.

Precautions

1. This product is intended solely for scientific research and must not be used for clinical diagnosis, treatment, or any other purposes.

2. For your safety and health, please wear a lab coat and disposable gloves when performing this procedure.

3. Reagents should be stored in a cool, dry, and well-ventilated environment.

4. This product can be used in conjunction with Eosin Staining Solution (Catalog No.: AS11L031).

5. When using this product for the first time, it is recommended to prepare 1–2 samples for a preliminary experiment.

Related Product Recommendations

General-purpose tissue fixation solution (4% paraformaldehyde) (Catalog No.: AC28L112)

Antifade reagent (Catalog No.: AC28L512)

Hematoxylin and Eosin (HE) Staining Kit (Catalog No.: AS11L011)

Eosin Staining Solution (Catalog No.: AS11L031)

This product is intended solely for scientific research and must not be used for clinical diagnosis, treatment, or any other purposes.