Hematoxylin And Eosin(HE) Staining Kit

Procedure: After freezing section, various steps (drying, fixation with ethanol or methanol, thermal fixation, use of charged or sticky cover) ensure that the tissue section does not fall off during dyeing. 1.95% ethanol: The tissue sections need to be fixed and hydrated before HE staining begins. The sections were placed in 95% ethanol for about 2min to begin hydration and fixation. Note: Skipping the use of 95% ethanol for tissue hydration and fixation does not affect morphological details. The logical explanation is that the second step will provide enough water for hydration. 2. Hydration: Further hydrate the tissue sections for 3-5 min, and flush out the remaining ethanol in the previous step. Note: Similar to step 1, skipping hydration has very little mass for tissue staining. 3. Hematoxylin: After hydration, the tissue sections were stained with hematoxylin for 1~3min, and the dyeing time could be adjusted according to the staining results and requirements. Hematoxylin is a basic dye that stains the acidic components of cells, including nucleic acids, mucopolysaccharides, and acidic glycoproteins, turning them blue-purple. Note: Without hematoxylin, eosin dyes the tissue bright pink; The obvious one is the lack of a nucleus. Tissue structure is difficult to distinguish, intracellular components are almost nonexistent, difficult to identify. 4. Washing: It is important to wash the tissue with water after hematoxylin staining. This step, followed by washing, removes excess dyes, mordants, etc. Proper rinsing can remove the metallic gloss on the surface, and the metallic gloss can prevent hematoxylin staining. Note: Skipping this step will result in undesirable results, such as formation of precipitates, dilution of dye, and presence of metallic gloss on the surface. 5 acid alcohol: acid alcohol color separation agent 3~5sec. In the hematoxylin staining method, where tissue sections are intentionally overstained, acid alcohol will remove excess dye and background staining on the slides. Note: Without this step, the tissue will turn dark purple. Acidophilic structures, such as collagen, will not appear bright pink. Organizational structures will be hard to tell apart. It's hard to make a correct judgment about the slice. Excessive color separation may be due to: 1) high acid concentration; 2) Decolorization time is too long. Insufficient color separation is due to: 1) acid concentration is too low; 2) decolorization time is insufficient; 3) Excess protein or gelatin on slices. 6. Water washing: Wash with water for 30sec to stop acid alcohol reaction and further prevent acid alcohol from removing a large amount of hematoxylin from tissue sections. Note: If the slices have not been rinsed, the sour alcohol can be excessively discolored. If the decolorizer is not removed, the acid alcohol will continue to remove the hematoxylin from the tissue sections. 7. Flush with tap water or blue solution: flush with tap water for 3~5min, can play blue treatment, purple hematoxylin into blue purple. In general, bluing reagents are pH dependent and consist of alkaline solutions. Therefore, if tap water is used as a blue reagent, daily pH monitoring is very important, as the pH of tap water may fluctuate daily. Or bluing solution bluing, 30sec~1min; Rinse with running tap water for 5min; Note: Skipping the blue reagent step will cause subtle changes in the color of the hematoxylin dye solution, which will be difficult to distinguish. Instead of dark blue, the nucleus will appear purple and sometimes even red. The nucleus should not be hypercyanosis. If the tissue is stained blue, it is usually due to too much hematoxylin in the tissue section. 8. Water washing: To prepare for eosin redyeing, wash with water for 30sec to remove the excess blue reagent. Because eosin does not stain in alkaline environments, washing the blue removal reagent will allow eosin to retain its acidic pH, allowing even redyeing. Note: Tissue sections cannot be stained correctly with eosin without washing after the blue reagent. As a result, acidic structures will be stained pale and uneven, leading to misjudgments. 9. Eosin: Eosin is an excellent hematoxylin redye for the proper differentiation of intracellular structures. Eosin redyeing 30~60sec, dyeing time can be adjusted according to dyeing results and requirements. Eosin is an acid dye that can stain cytoplasm, especially mitochondria, secretory granules, and collagen. It can distinguish between the different organelles in the cell and the different connective tissues. Note: Failure to use eosin to restain tissue sections will result in poorly differentiated tissue sections. Weak staining is due to: 1) thin tissue sections; 2) Insufficient dyeing time; 3) Subsequently 95% of the alcohol was hyperdifferentiated. Excessive dyeing is due to: 1) high concentration of dyeing solution (possibly due to excessive evaporation); 2) Too thick tissue section; 3) Too long dyeing time; 4) Subsequently 95% of the alcohol was underdifferentiated. Undifferentiated eosin staining (one color) is due to: 1) incomplete fixation; 2) overstaining, a) too long dyeing time; B) Excessive concentration of dye solution. 10.95% ethanol: 95% ethanol treatment for 2 s~1 min, according to the dyeing results and requirements; Eosin differentiation will produce different pinks. Note: Differentiation is less effective if 95% ethanol is diluted (e.g., with eosin from a previous stain). 11.100% ethanol: Decolorize tissue sections by 100% ethanol. Treated with 100% ethanol for 1 min; Then treat with the new 100% ethanol for 1 min. Note: This step is difficult to notice. Without 95% ethanol, it is difficult to distinguish acidic structures and the tissue sections turn pink. Without 100% ethanol dehydration, the tissue sections could not be completely dehydrated, resulting in the formation of water droplets under the cover. These droplets can combine with the next step of xylene to form a white haze, which will affect the quality of the tissue section. 12. Xylene: Treated with xylene for 5min. After sufficient dehydration, xylene removes alcohol from tissue sections. Xylene can also be used as an embedding agent to ensure dissolution when the slide is capped after the removal of liquor. Because of their potential toxicity, xylene substitutes such as limonene and cycloalkanes may also be used. Alternatives offer the same quality of tissue staining, and they are safer to use and easier to operate. Note: When skipping this step without xylene transparency, the alcohol will be preserved on the tissue section, causing eosin to flow out of the section. 13. Sealing: neutral gum sealing, natural air drying or baked dry, microscope observation.

HE dyeing is a dyeing with high requirements for experience. The dyeing time provided in the experimental procedure is all reference time, which should be determined according to the experimental principle and specific conditions.

Store at room temperature, dark for 12 months.