一、实验器材及试剂

1、实验器材

2、主要试剂及货号

二、实验步骤

1、组织切片常规脱蜡至水，将脱好水的切片浸泡于Bouin氏液或Zenker氏液过夜，流水冲洗干净。

2、Harris苏木素或铁苏木素染色5-10分钟，流水稍洗。

3、用0.8%-1%盐酸酒精分化，流水冲洗数分钟；碳酸锂返蓝数秒，流水冲洗。

4、丽春红酸性品红染液染5-10分钟，流水稍冲洗。

5、磷钼酸溶液处理约5分钟，不用水洗，直接用苯胺蓝染液复染5分钟。

6、1%冰醋酸处理1分钟，95%酒精脱水多次。

7、无水酒精脱水，二甲苯透明，中性树胶封固。

三、结果判读

胶原纤维呈蓝色（用苯胺蓝液复染）或绿色（用亮绿复染）。胞质、肌纤维和红细胞呈红色。细胞核呈蓝褐色。

四、注意事项

1、组织用Bouin氏液或Zenker氏液固定为佳。若已用10%甲醛液固定，切片可在脱蜡至水后，再放入Bouin氏液常温作用一晚或置37℃温箱内1-2小时，然后流水冲洗切片至黄色消失再进行染色。

2、磷钼酸处理时需要镜下控制，见肌纤维呈红色，胶原纤维呈淡红色即可。

3、冰醋酸分化过程中应严格控制时间，若分化过度，胶原蓝色太浅；若分化不足，易与红色叠加呈紫蓝色。

Experimental Report on Masson Staining

1.Instruments and key reagents

1.1Instruments

1.2Key reagents

2.Procedures

2.12.1Deparaffinization and rehydration.

2.1.1Tissue sections were immersed in clearer for 10min. Repeat this step two times, gently shaking off excess liquid between each step.

2.1.2Tissue sections were immersed in progressively more dilute ethanol solutions and ultimately immersed in distilled water to rehydrate the tissue: Absolute ethanol for 5min, 95% ethanol for 5min, 85% ethanol for 5min, 75% ethanol for 5min. Rinsing with distilled water for 1min.

2.1.3The dehydrated tissue sections were immersed in Bouin’s solution or Zenker’s solution overnight, then, rinsed with running water.

2.2Sections were stained with hematoxylin solution (Harris) or iron hematoxylin for 5-10 minutes and slightly washed with running water.

2.3Sections were differentiated with 0.8% - 1% hydrochloric acid alcohol and washed with running water for several minutes. Sections can also be treated with lithium carbonate solution to be bluer and washed with running water.

2.4Sections were stained with ponceau acid fuchsin solution for 5-10 minutes and washed with running water.

2.52.5Sections were treated with phosphomolybidic acid solution for about 5 minutes and then stained with aniline blue solution for 5 minutes without washing.

2.6Sections were treated with 1% glacial acetic acid for 1 minute and dehydrated with 95% alcohol for several times.

2.7The tissue sections were dehydrated with absolute alcohol and transparent with xylene, then mounted with neutral balsam. Examination with microscope.

3.Results

TCollagen fibers were blue (counterstained with aniline blue solution) or green (counterstained with bright green solution). The cytoplasm, muscle fibers and red blood cells are red. Nuclei are blue brown.

4.Attentions

4.1It is better to fix the tissue with Bouin’s solution or Zenker’s solution. If it has been fixed with 10% formaldehyde solution, the tissue sections can be dewaxed to water, and then put into Bouin’s solution overnight at room temperature or put into a 37℃ incubator for 1-2 hours. Then, washed sections with running water until the yellow disappeared before staining.

4.2It need to be controlled under microscope during treatment of phosphomolybdic acid. It can be seen that muscle fibers are red and collagen fibers are light red.

4.3It is important to control the time of differentiation with glacial acetic acid, if the differentiation is excessive, the blue of collagen will be too light. If the differentiation is insufficient, it is easy to overlap with red and become purple blue.

5.Technical support

The technical support of Masson staining experiment is provided by Hubei BIOSSCI Biotech Co., Ltd (Wuhan Changyan Pathology technology Co., Ltd).。

Tel: 400 118 0100

Fax: +86-027-87382710

E-mail: support@biossci.com

Website: www.biossci.com