髓鞘染色操作步骤5.9.doc

髓鞘染色操作步骤Reagent：Luxol fast blue MBS（solvent blue 38），Alcohol，Lithium carbonate，Glacial acetic acid，试剂：Luxol 固蓝 MBS（solvent blue 38），乙醇，碳酸锂，冰醋酸，Section：15-25 um切片：15-25umsolutions溶液1 0.1% Luxol fast blue solution: 1 0.1% 固蓝溶液Luxol fast blue MBS（solvent blue 38） 0.1 g 固蓝 0.1 gEthyl Alcohol，95% 100 ml 95% 乙醇 100 mlGlacial acetic acid 0.02 ml 冰醋酸 0.02 ml取固蓝0.1g 加入95%乙醇，在温度50-60条件下，于磁力搅拌器中搅拌，用10冰乙酸调PH至4.3。2 0.05% Lithium carbonate solution: 2 0.05%碳酸锂溶液Lithium carbonate 0.05 g碳酸锂 0.05 gDistilled water 100 ml去离子水 100 ml充分混均。备注：此液需经常更换。3 0.1%cresyl violet acetate solution 0.1%甲苯粉紫溶液 cresyl violet acetate 0.1g 甲苯粉紫 0.1g Distilled water 100ml 去离子水 100mLAcetic acid 0.1mL冰乙酸 0.1mLMeasure 0.1g cresyl violet acetate ，add to 100mL distilled water and stir until it is dissolved，add to 0.1mL acetic acid make PH to 3.7.This solution should be prepared 16-20h prior to use.称取0.1甲苯粉紫醋酸盐粉末，加100ml去离子水搅拌至完全溶解，加入0.1ml冰乙酸，调节ph至3.7。该溶液需在16-20小时前配制。Staining procedure for frozen section冰冻切片染色步骤1. section Place in 70% ethanol for approximately 10-15 min.取出切片，于70%乙醇溶液中浸泡10-15 min。 2. Incubate in Luxol fast blue MBS solution for over night（16-24 h）at 57.取出切片，置于Luxol固蓝溶液中，在57温度下，孵育过夜（16-24 h）。目的：使髓鞘着色。3. section rinse in 95% ethanol。 取出切片，于95%乙醇溶液中浸洗。目的：洗去过多染液。4. rinse in distilled water.取出切片，于去离子水中浸洗。5. Begin differentiation by quick immersion in a 0.05% aqueous lithium carbonate solution for 3-10 seconds.切片于0.05%碳酸锂溶液中快速浸洗3-10s。6. Differentiate in 70% ethanol. Until gray and white matte can be distinguished .care should be taken not to over differentiate.立即取出切片，置于70%乙醇溶液中分化，直到灰质和白质能够清晰辨别。7. wash in distilled water.取出切片，于去离子水中冲洗。8. finish the differentiation by briefly rinsing in 0.05% lithium carbonate and then putting through several changes of 70% alcohol until there is a sharp contrast between the greenish-blue color of the white matter and the colorless gray matter. Care should be taken to rinse only briefly in the lithium carbonate（3-5 second）since the final most delicate differentiation occurs in 70% alcohol.切片于0.05%碳酸锂溶液中轻轻浸洗数次，然后置于70%乙醇溶液中浸洗数次，直到白质颜色转变成蓝绿色，灰质几无颜色时，停止分化。9. wash thoroughly in distilled water.切片去离子水中冲洗完全。10. section place in 1%solution of cresyl violet acetate at 57，stain for 2 minutes（the solution should be preheated）。 切片入1%甲苯粉紫溶液，于57下，浸染2min （溶液需提前预热） 11. section Place in 95% ethanol for approximately 1 min.切片于95%乙醇溶液中浸泡1 min。12. section Place in 95% ethanol for approximately 3 min.切片于95%乙醇溶液中浸泡3 min。13. section Place in 100% ethanol for approximately 3 min.切片于100%乙醇溶液中浸泡3 min。14. immersed in xylene for 5min and coversliped with D.P.X.二甲苯透明5 min。中性树胶封固。15. 镜下观察髓鞘。Result：The myelin, including phospholipids, will be stained blue to green,结果：髓鞘：蓝绿色。参考文献1 Kluver H, Barrera E. A method for the combined staining of cells and fibers in the nervous systemJ. J Neuropathol Exp Neurol, 1953, 12(4): 400-3.2 Myelinated