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。
Cole苏木素染色液是一种化学氧化成熟的明矾苏木素,主要由苏木精、硫酸铝钾、甘油等组成。染色液中苏木精含量小,无氧化膜形成,对细胞核染色很清晰,不着染细胞质和纤维成分,属进行性染色,故染色后可以不用盐酸乙醇分化,染色时间约5~10min,主要用于复染细胞核。
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
2、染色
①Cole苏木素染色液:5-10min。
②伊红染色液染色:3-5min。
③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、Cole苏木素染色液滴染1-2min(可加热至50℃)。
4、自来水冲洗:5-10s。
5、伊红染色液:2-5s。
6、自来水冲洗:1-2s。
7、80%乙醇:1-2s。
8、95%的乙醇:1-2s。
9、无水乙醇:2-5s。
10、苯酚二甲苯(1:3):2-5s。
11、二甲苯透明3次,每次2-5s。
12、中性树脂封片。
染色结果:细胞核呈蓝色;细胞质、纤维呈红色。
(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;角蛋白,红细胞等呈明亮的橙红色。
注意事项:
1、切片脱蜡应尽量干净。 系列乙醇应经常更换新液。
2、冷冻切片染色时间尽量要短。
3、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。
4、为了您的安全和健康,请穿实验服并戴一次性手套操作。
5、本产品仅供科研使用,严禁它用。
Hematoxylin-Eosin staining, commonly referred to as HE staining, is the most widely used staining method in pathology and histology. Hematoxylin is an alkaline natural dye that stains 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 face outward, making the outer side of the DNA double helix negatively charged and acidic. This allows it to easily bind to the positively charged alkaline hematoxylin dye via ionic or hydrogen bonds, resulting in staining.
Cole Hematoxylin Staining Solution is a chemically oxidized and mature alum hematoxylin, mainly composed of hematoxylin, potassium aluminum sulfate, glycerol, and other components. It contains a low concentration of hematoxylin and does not form an oxide film. It stains cell nuclei clearly without coloring the cytoplasm or fibrous components, and belongs to progressive staining—thus, hydrochloric acid-ethanol differentiation is not required after staining. The staining time is approximately 5-10 minutes, and it is mainly used for counterstaining cell nuclei.
Materials to Be Prepared by the User
1. Hydrochloric acid-ethanol differentiation solution
2. Bluing solution (e.g., dilute ammonia water, lithium carbonate solution, etc.)
3. Graded ethanol
4. Eosin staining solution
5. 4% paraformaldehyde
Operating Procedures (For Reference Only)
(1) Staining of Paraffin Sections
1. Deparaffinize sections to water
2. Staining
① Cole Hematoxylin Staining Solution: 5-10 minutes.
② Eosin Staining Solution: 3-5 minutes.
③ Rinse with tap water: 1-5 seconds.
3. Dehydration, Clearing, and Mounting
① 80% ethanol: 10-20 seconds.
② 90% ethanol: 10-20 seconds.
③ 95% ethanol: 2 applications, 1-2 minutes each.
④ Absolute ethanol: 2 applications, 2-3 minutes each.
⑤ Xylene clearing: 3 applications, 2-3 minutes each.
⑥ Mount sections with neutral balsam.
Staining Results: Cell nuclei appear blue; cytoplasm, muscle fibers, collagen fibers, etc., appear red in varying shades; keratin, red blood cells, etc., appear bright orange-red.
(2) Staining of Frozen Sections
1. Ethyl ether-ethanol mixed fixative for 5-10 seconds.
2. Rinse with tap water for 2-5 seconds.
3. Dropwise staining with Cole Hematoxylin Staining Solution for 1-2 minutes (may be heated to 50°C).
4. Rinse with tap water for 5-10 seconds.
5. Eosin Staining Solution for 2-5 seconds.
6. Rinse with tap water for 1-2 seconds.
7. 80% ethanol for 1-2 seconds.
8. 95% ethanol for 1-2 seconds.
9. Absolute ethanol for 2-5 seconds.
10. Phenol-xylene (1:3) for 2-5 seconds.
11. Xylene clearing for 3 applications, 2-5 seconds each.
12. Mount sections with neutral balsam.
Staining Results: Cell nuclei appear blue; cytoplasm and fibers appear red.
(3) Cell Staining
1. Fixation with 4% paraformaldehyde: 10-20 minutes.
2. Rinse with tap water: 2 applications, 2 minutes each.
3. Rinse with distilled water: 2 applications, 2 minutes each.
4. Follow the same staining, deparaffinization, clearing, and mounting steps as for paraffin sections, but reduce the reaction time accordingly.
Staining Results: Cell nuclei appear blue; cytoplasm and fibers appear red; keratin, red blood cells, etc., appear bright orange-red.
Precautions
1. Ensure thorough deparaffinization of sections. Replace serial ethanol solutions with fresh ones regularly.
2. Minimize the staining time for frozen sections.
3. Common bluing solutions include 0.2-1% ammonia water, Scott’s bluing solution, or 0.1-1% lithium carbonate solution.
4. For your safety and health, wear a lab coat and disposable gloves during operation.
5. This product is for scientific research use only. Any other uses are strictly prohibited.