髓鞘染色操作步骤5.9

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1、髓鞘染色操作步骤Reagent：LuxolfastblueMBS（solventblue38），Alcohol，Lithiumcarbonate，Glacialaceticacid，试剂：Luxol固蓝MBS（solventblue38），乙醇，碳酸锂，冰醋酸，Section：15-25um切片：15-25umsolutions溶液10.1%Luxolfastbluesolution:10.1%固蓝溶液LuxolfastblueMBS（solventblue38）0.1g固蓝0.1gEthylAlcohol，95%

2、100ml95%乙醇100mlGlacialaceticacid0.02ml冰醋酸0.02ml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。20.05%Lithiumcarbonatesolution:20.05%碳酸锂溶液Lithiumcarbonate0.05g碳酸锂0.05gDistilledwater100ml去离子水100ml充分混均。备注：此液需经常更换。30.1%cresylvioletacetatesolution0.1%甲苯粉紫溶液cres

3、ylvioletacetate0.1g甲苯粉紫0.1gDistilledwater100ml去离子水100mLAceticacid0.1mL冰乙酸0.1mLMeasure0.1gcresylvioletacetate，addto100mLdistilledwaterandstiruntilitisdissolved，addto0.1mLaceticacidmakePHto3.7.Thissolutionshouldbeprepared16-20hpriortouse.称取0.1甲苯粉紫醋酸盐粉末，加100ml去离子

4、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。Stainingprocedureforfrozensection冰冻切片染色步骤1.sectionPlacein70%ethanolforapproximately10-15min.取出切片，于70%乙醇溶液中浸泡10-15min。2.IncubateinLuxolfastblueMBSsolutionforovernight（16-24h）at57℃.取出切片，置于Luxol固蓝溶液中，在57℃温度下，孵育过夜（16-24

5、h）。目的：使髓鞘着色。3.sectionrinsein95%ethanol。取出切片，于95%乙醇溶液中浸洗。目的：洗去过多染液。4.rinseindistilledwater.取出切片，于去离子水中浸洗。5.Begindifferentiationbyquickimmersionina0.05%aqueouslithiumcarbonatesolutionfor3-10seconds.切片于0.05%碳酸锂溶液中快速浸洗3-10s。6.Differentiatein70%ethanol.Untilgrayand

6、whitemattecanbedistinguished.careshouldbetakennottooverdifferentiate.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.washindistilledwater.取出切片，于去离子水中冲洗。8.finishthedifferentiationbybrieflyrinsingin0.05%lithiumcarbonateandthenputtingthroughseveralchangesof70%alcoholuntilthe

7、reisasharpcontrastbetweenthegreenish-bluecolorofthewhitematterandthecolorlessgraymatter.Careshouldbetakentorinseonlybrieflyinthelithiumcarbonate（3-5second）sincethefinalmostdelicatedifferentiationoccursin70%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

8、成蓝绿色，灰质几无颜色时，停止分化。1.washthoroughlyindistilledwater.切片去离子水中冲洗完全。2.sectionplacein1%solutionofcresylvioletacetateat57℃，stainfor2minutes（thesolutionshouldbepreheated）。切片入1%甲苯粉紫溶液，于57℃下