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改良Harris苏木素染色液
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BioReagent,用于显微镜,适用于微生物学 级 ，适用于对基线干扰要求严格的色谱和分析工作流程。
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文献证明
在色谱分析、有机合成和交叉偶联反应领域已被 0 篇同行评审文献引用。
概述
苏木素(Hematoxylin)和伊红(Eosin)联合染色简称HE染色，是病理学和组织学最常用的一种染色方法。苏木精为碱性天然染料，可使细胞核着色,细胞核内染色质的主要成分是DNA，在DNA的双螺旋结构中，两条核苷酸链上的磷酸基向外，使DNA双螺旋的外侧带负电荷，呈酸性，很容易与带正电荷的苏木精碱性染料以离子键或氢键结合而被染色。
改良Harris苏木素染色液是最经典的Harris苏木素染色法的改进，该染色液不含汞，无毒，无氧化膜，细胞核染色质着色深而细微，临床上常替代Harris苏木素染色液，染色时间一般5～8min，其特点是苏木素被氧化的程度高，染色力强，虽然染色时间短，但是易使细胞核、细胞质、纤维过染，染色后需要盐酸乙醇分化，属于退行性染色。
染色原理：
1、细胞核染色原理：苏木素为碱性天然染料，可使细胞核着色，细胞核内染色质的成分主要是DNA，在DNA双螺旋结构中两条核苷酸链上的磷酸基向外，使DNA双螺旋的外侧带负电荷，呈酸性，很容易与带正电荷的苏木素碱性染料以离子键或氢键结合而被染色。苏木素在碱性溶液中呈蓝色，所以细胞核被染成蓝色。
2、细胞浆染色原理：伊红是一种化学合成的酸性染料，在一定条件下可使细胞浆着色，细胞浆的主要成分是蛋白质，为两性化合物，细胞浆的染色与染液的pH值密切相关，当染色液pH值在胞浆蛋白质等电点(4.7～5.0)以下时，胞浆蛋白质以碱式电离，则细胞浆带正电荷，就可被带负电荷的酸性染料染色。伊红在水中离解成带负电荷的阴离子，与胞浆蛋白质带正电荷的阳离子结合，使细胞浆着色，呈现红色。
3、分化作用：染色后，用某些特定的溶液将组织过多结合的染色剂脱去，这个过程称为分化作用，所用的溶液称为分化液。在HE染色中常用0.5－1%盐酸乙醇作为分化液，因酸能破坏苏木素的醌型结构，使组织与色素分离而退色，大多数组织经苏木素染色后，必须用盐酸乙醇分化，使细胞核过多结合的苏木素染料和细胞浆吸附的苏木素染料脱去，再进行伊红染色，才能保证细胞核与细胞浆染色的分明。
4、返蓝作用：分化之后，苏木素在酸性条件下处于红色离子状态，呈红色；在碱性条件下处于蓝色离子状态，呈蓝色。组织切片经酸性乙醇分化后呈红色或粉红色，立即用水除去组织切片上的酸而中止分化，再用弱碱性水使苏木素染上的细胞核呈现蓝色，这个过程称为返蓝作用或蓝化作用，另外用自来水(尤其是温水)浸洗也可使细胞核返蓝，但所需时间较长。
自备材料：
1、盐酸乙醇分化液
2、蓝化液，如稀氨水、碳酸锂溶液等
3、系列乙醇
4、伊红染色液
5、4%多聚甲醛
操作步骤(仅供参考)：
(一)石蜡切片染色
1、 切片脱蜡至水
①二甲苯作用2次，每次5-10min。
②(可选)无水乙醇作用2次，每次3-5min。
③95%的乙醇：3-5min
④90%的乙醇：3-5min
⑤80%的乙醇：3-5min
⑥自来水或蒸馏水冲洗：1-3min
2、染色
①改良Harris苏木素染色液染色：3-8min
②自来水或蒸馏水冲洗：5-10s
③(可选)盐酸乙醇分化：2-5s
④自来水冲洗：20-30s
⑤(可选)蓝化液返蓝：20-40s
⑥自来水冲洗：30-60s
⑦伊红染色液(水溶)染色：0.5-2min
⑧自来水冲洗：1-5s
3、脱水、透明、封固
①80%乙醇：10-20s
②90%乙醇：10-20s
③95%乙醇作用2次，每次1-2min。
④无水乙醇作用2次，每次2-3min。
⑤二甲苯透明3次，每次2-3min。
⑥中性树脂封片。
染色结果：
细胞核呈蓝色。
细胞质、肌纤维、胶原纤维等呈深浅不一的红色。
角蛋白、红细胞等呈明亮的橙红色。
(二)冰冻切片染色
1、乙醚-乙醇混合固定液：5-10s
2、自来水冲洗：2-5s
3、改良Harris苏木素染色液滴染1-2min(可加热至50℃)
4、自来水冲洗：2-5s
5、(可选)盐酸乙醇分化：2-5s
6、自来水冲洗：2-5s
7、蓝化液返蓝或温水返蓝：2-5s
8、 80%乙醇脱水5-10s
9、伊红染色液染色：2-5s
10、 80%的乙醇：1-2s
11、95%的乙醇：1-2s
12、无水乙醇：2-5s
13、苯酚二甲苯(1:3)：2-5s
14、二甲苯透明3次，每次2-5s。
15、中性树脂封片
(三)细胞染色
1、4%多聚甲醛固定10-20min。
2、自来水冲洗2次，每次2min。
3、蒸馏水冲洗2次，每次2min。
4、染色、脱蜡、透明、封固步骤同石蜡切片的染色步骤，作用时间应相应缩短。
染色结果：
细胞核呈蓝色。
细胞质、纤维呈红色。
注意事项：
1、 切片脱蜡应尽量干净。
2、 系列乙醇应经常更换新液。
3、 盐酸乙醇分化时间应根据切片厚薄、组织类别以及新旧而定，另外分化后自来水冲洗时间应该足够，以便彻底清洗酸。
4、 乙醚-乙醇混合固定液是由乙醚和95%乙醇等量混合而得，再加入适量乙酸，密闭保存。
5、 冷冻切片染色时间尽量要短。
6、 蓝化液常使用0.2～1%氨水或Scott促蓝液或0.1～1%碳酸锂溶液。
7、为了您的安全和健康，请穿实验服并戴一次性手套操作。
8、本产品仅供科研使用，严禁它用。
The combined staining of Hematoxylin and Eosin, referred to as HE staining, is the most commonly used staining method in pathology and histology. Hematoxylin is a basic natural dye that can stain cell nuclei. The main component of chromatin in the nucleus is DNA. In the double-helix structure of DNA, the phosphate groups on the two nucleotide chains are outwardly oriented, making the outer side of the DNA double helix negatively charged and acidic. It easily binds to the positively charged basic hematoxylin dye through ionic bonds or hydrogen bonds and is thus stained.
Improved Harris Hematoxylin Staining Solution is an improvement on the classic Harris hematoxylin staining method. This staining solution is mercury-free, non-toxic, and free of oxide film. It stains nuclear chromatin deeply and delicately, and is often used clinically as a substitute for Harris hematoxylin staining solution. The staining time is generally 5-8 minutes. Its characteristics include a high degree of oxidation of hematoxylin and strong staining power. Although the staining time is short, it tends to overstain cell nuclei, cytoplasm, and fibers. After staining, hydrochloric acid-ethanol differentiation is required, making it a regressive staining method. This reagent is for research use only and not suitable for clinical diagnosis or other purposes.
Staining Principles
1. Nuclear Staining Principle: Hematoxylin is a basic natural dye capable of staining cell nuclei. The main component of chromatin in the nucleus is DNA. In the double-helix structure of DNA, the phosphate groups on the two nucleotide chains are outwardly directed, rendering the outer side of the DNA double helix negatively charged and acidic. It readily binds to the positively charged basic hematoxylin dye via ionic or hydrogen bonds, resulting in staining. Hematoxylin appears blue in alkaline solutions, thus staining the cell nucleus blue.
2. Cytoplasmic Staining Principle: Eosin is a chemically synthesized acidic dye that can stain cytoplasm under certain conditions. The main component of cytoplasm is protein, which is an amphoteric compound. The staining of cytoplasm is closely related to the pH value of the staining solution. When the pH value of the staining solution is below the isoelectric point (4.7-5.0) of cytoplasmic proteins, the cytoplasmic proteins ionize in a basic form, making the cytoplasm positively charged, which can then be stained by the negatively charged acidic dye. Eosin dissociates into negatively charged anions in water, which combine with the positively charged cations of cytoplasmic proteins to stain the cytoplasm red.
3. Differentiation: After staining, the process of removing excess bound stain from the tissue using specific solutions is called differentiation, and the solution used is called a differentiating solution. In HE staining, 0.5-1% hydrochloric acid-ethanol is commonly used as the differentiating solution. Acids can destroy the quinone structure of hematoxylin, causing the separation of tissue and pigment and subsequent decolorization. After hematoxylin staining, most tissues must undergo hydrochloric acid-ethanol differentiation to remove excess hematoxylin dye bound to the nucleus and hematoxylin dye adsorbed by the cytoplasm before eosin staining. This ensures clear differentiation between nuclear and cytoplasmic staining.
4. Blueing: After differentiation, hematoxylin exists in a red ionic state under acidic conditions (appearing red) and in a blue ionic state under alkaline conditions (appearing blue). Tissue sections stained red or pink after acidic ethanol differentiation must be immediately rinsed with water to stop differentiation, followed by treatment with weakly alkaline water to make the hematoxylin-stained nuclei appear blue. This process is called blueing. Alternatively, rinsing with tap water (especially warm water) can also achieve nuclear blueing, but it requires a longer time.
Materials to Be Prepared by the User
1. Hydrochloric acid-ethanol differentiating solution.
2. Blueing solution, such as dilute ammonia water, lithium carbonate solution, etc.
3. Graded ethanol.
4. Eosin staining solution.
5. 4% paraformaldehyde.
Operation Steps (For Reference Only)
(1) Paraffin Section Staining
1. Deparaffinization to Water
① Treat with xylene or Leagene Deparaffinization and Clearing Solution twice, 5-10 minutes each time.
② (Optional) Treat with absolute ethanol twice, 3-5 minutes each time.
③ 95% ethanol for 3-5 minutes.
④ 90% ethanol for 3-5 minutes.
⑤ 80% ethanol for 3-5 minutes.
⑥ Rinse with tap water or distilled water for 1-3 minutes.
2. Staining
① Stain with Improved Harris Hematoxylin Staining Solution for 3-8 minutes.
② Rinse with tap water or distilled water for 5-10 seconds.
③ (Optional) Differentiate with hydrochloric acid-ethanol for 2-5 seconds.
④ Rinse with tap water for 20-30 seconds.
⑤ (Optional) Blue with blueing solution for 20-40 seconds.
⑥ Rinse with tap water for 30-60 seconds.
⑦ Stain with eosin staining solution (water-soluble) for 0.5-2 minutes.
⑧ Rinse with tap water for 1-5 seconds.
3. Dehydration, Clearing, and Mounting
① 80% ethanol for 10-20 seconds.
② 90% ethanol for 10-20 seconds.
③ Treat with 95% ethanol twice, 1-2 minutes each time.
④ Treat with absolute ethanol twice, 2-3 minutes each time.
⑤ Clear with xylene three times, 2-3 minutes each time.
⑥ Mount with neutral resin.
Staining Results
Cell nuclei: blue.
Cytoplasm, muscle fibers, collagen fibers, etc.: red of varying intensities.
Keratin, red blood cells, etc.: bright orange-red.
(2) Frozen Section Staining
1. Fix with ether-ethanol mixed fixative for 5-10 seconds.
2. Rinse with tap water for 2-5 seconds.
3. Drop-stain with Improved Harris Hematoxylin Staining Solution for 1-2 minutes (may be heated to 50℃).
4. Rinse with tap water for 2-5 seconds.
5. (Optional) Differentiate with hydrochloric acid-ethanol for 2-5 seconds.
6. Rinse with tap water for 2-5 seconds.
7. Blue with blueing solution or warm water for 2-5 seconds.
8. Dehydration with 80% ethanol for 5-10 seconds
9. Stain with eosin staining solution for 2-5 seconds.
10. 80% ethanol for 1-2 seconds.
11. 95% ethanol for 1-2 seconds.
12. Absolute ethanol for 2-5 seconds.
13. Phenol-xylene (1:3) for 2-5 seconds.
14. Clear with xylene three times, 2-5 seconds each time.
15. Mount with neutral resin.
(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, but reduce the reaction time accordingly.
Staining Results
Cell nuclei: blue.
Cytoplasm, fibers: red.
Precautions
1. Ensure thorough deparaffinization of sections.
2. Regularly replace graded ethanol with fresh solutions.
3. The differentiation time with hydrochloric acid-ethanol depends on section thickness, tissue type, and reagent freshness. Additionally, rinse with tap water sufficiently after differentiation to completely remove 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 in a sealed container.
5. Minimize staining time for frozen sections.
6. Common blueing solutions include 0.2-1% ammonia water, Scott's blueing solution, or 0.1-1% lithium carbonate solution.
7. For your safety and health, wear a lab coat and disposable gloves during operation.
8. This product is for scientific research use only. Any other uses are strictly prohibited.
规格
英文别名
Modified Harris' Hematoxylin Stain Solution
规格或纯度
BioReagent,适用于微生物学,用于显微镜
稳定性与储存
Store at room temperature long term (12 months). Store in the dark.
英文名称
Improved Harris Hematoxylin Staining Solution
储存条件
避光,室温
运输条件
常规运输
产品线
生物产品
名称和识别符
分子类型
生物试剂/缓冲液
技术文档
高级数据
化学和物理性质
敏感性
Light-sensitive
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| 批号(Lot Number) | 证书类型 | 货号 |
|---|---|---|
 ZJ25F1230701 |
分析证书 | I1507737 |
| ZJ25F1230702 |
分析证书 | I1507737 |
技术文档和文章
溶液计算器
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本产品按常温标准条件运输，无需温控包装。
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