卢卡斯快蓝染色方法.doc

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1、Description: Thisisforstainingofmyelin/myelinatedaxonsonformalin-fixed,paraffin-embeddedbrainandspinalcordtissuesections,aswellasfrozensections.Themyelin,includingphospholipids,willbestainedbluetogreen,andtheneuronswillbestainedviolet.Thisstainiscommonlyusedforidentifyingthebasicneuronalstructurei

2、nbrainorspinalcordtissue. Fixation: 10%formalin. Section: paraffinsectionsat5-10um.Frozensectionsat20-30um. SolutionsandReagents: 0.1%Luxolfastbluesolution:      Luxolfastblue,MBS--------------------------0.1gm      EthylAlcohol,95%----------------------------100ml      Glacialaceticacid----------

3、------------------0.5ml 0.1%Cresylechtvioletsolution:      Cresylechtviolet(cresylfastviolet)-------0.1gm      Distilledwater--------------------------------100ml      Add10dropsofglacialaceticacidjustbeforeuseandfilter. 0.05%Lithiumcarbonatesolution:      Lithiumcarbonate-------------------------

4、------0.05gm      Distilledwater-----------------------------------100ml Procedure:1.   Deparaffinizeandhydrateto95%ethylalcohol(Frozen/vibratomesectionsmayneedtohavede-fatstep:placesectionsdirectlyinto1:1alcohol/chloroformforafewhours/overnightandthenhydrateback95%ethylalcohol).2.Leaveinluxolfast

5、bluesolutionin56Covenovernight(forfrozensections,notlongerthan16hours).3.   Rinseoffexcessstainwith95%ethylalcohol.4.   Rinseindistilledwater.5.Differentiatetheslidesinthelithiumcarbonatesolutionfor30seconds.6.   Continuedifferentiationinthe70%ethylalcoholfor30seconds.7.Rinseindistilledwater.8.Che

6、ckmicroscopicallytoseeifgraymatterisclearandwhitemattersharplydefined.9.   Repeatthedifferentiationsteps(step5-7)ifnecessary.10.Whendifferentiationiscomplete,placeindistilledwater.11. Counterstaininthecresylvioletsolutionfor30-40seconds.12.Rinseindistilledwater(don’trinsein70%alcoholwhichwillclear

7、offfastbluestaining).13.Differentiatetheslidesin95%ethylalcoholfor5minutes(checkmicroscopically).14.100%alcohol2x5min.15.Xylene2x5min.16.Mountwithresinousmedium. Results:     Myelin,includingphospholipids--------