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OilRedOStainingProtocolDescription:Thisprotocolisforlipidandfatstainingonfrozensections.Itmaynotsuitableforparaffinembeddedtissuesections.Fixation:10%formalin(fixationisnecessary,damageofunfixedsectionshasbeenseen)Section:Frozensectionsat5-10umthickSolutionsandReagents:0.5%OilRedOSolution:OilRedO--------------------------0.5gPropyleneglycol,100%-----------100ml(Propyleneglycolisalsocalled1,2-Propanediol,SigmaCat#398039-500MLorCat#P4347)AddasmallamountofpropyleneglycoltotheoilredOandmixwell,crushlargerpieceswithstirringbar.Graduallyaddtheremainderofthepropyleneglycolwhilestirring.Heatgentlyuntilthesolutionreaches95-100ºC.Donotallowtogoover110ºC.Overheatingwillresultinhighbackgroundstaining.Filtersolutionthroughcoarsefilterpaper(i.e.25umfilterpaper)whilestillwarm.Allowtostandovernightatroomtemperature.Thesolutioncanbestoredatroomtemperatureformanyyears.Ifprecipitateformsinthesolution,re-filter.85%PropyleneGlycolSolution:Propyleneglycol,100%-----------------85mlDistilledwater--------------------------15mlGill'sorMayer'sHamatoxylinSolutionProcedure:Cutfreshfrozentissuesectionsat5-10umthickandmountonslides.Airdryslidesfor30-60minutesatroomtemperatureandthenfixinicecold10%formalinfor5-10minutes.Airdryagainforanother30-60minutesorrinseimmediatelyin3changesofdistilledwater.Letslidesairdryforafewminutes.Placeinabsolutepropyleneglycolfor2-5minutestoavoidcarryingwaterintoOilRedO.Staininpre-warmedOilRedOsolutionfor8-10minutesin60ºCoven.Differentiatein85%propyleneglycolsolutionfor2-5minutes.Rinsein2changesofdistilledwater.StaininGill'sorMayer'shamatoxylinfor30seconds.Washthoroughlyinrunningtapwaterfor3minutes.Placeslidesindistilledwater.Mountwithglycerinjellyorotheraqueousmountingmedium.Results:Lipids-----------------------------redNuclei-----------------------------paleblue成脂诱导及鉴定待细胞爬片基本融合后，加入含10-7mol/L地塞米松，5mg/L胰岛素，0.5mmol/L3-异丁甲基黄嘌呤，60μmol/L吲哚美辛，1O%FBS的DMEM成脂诱导液培养[3]，每3d换液，连续诱导21d，倒置显微镜下观察。油红O染色：取诱导21d细胞爬片PBS冲洗后加入10％中性甲醛，固定60min，吸去固定液，加入现配过滤后的OilRedO染色液，染色60min，用PBS洗3次，去处残留的染色液和残渣，显微镜下拍照。1.2.5成骨诱导及鉴定细胞爬片基本融合后，加入10-7mol/L地塞米松，0.15mmol/L维生素C，2mmol/Lβ-甘油磷酸钠，1O%FBS的DMEM成骨诱导液培养,每3d换液，连续诱导21d。茜素红染色：取诱导21d细胞爬片用10％中性甲醛固定30min，吸去固定液，加入0.1%的茜素红染色液，染色6-10min；用PBS洗两次，去处残留的染色液洗去未结合染料，甩干玻片后，甘油明胶封片。1.2.6成软骨细胞诱导及鉴定细胞爬片基本融合后，加入含100ng/ml生长分化因子5，10-7M地塞米松、50μg/ml维生素C、1％FBS、1%ITS+、40μg/mlL-脯氨酸和100μg/ml丙酮酸钠的DMEM高糖溶液培养[4]，每隔3d换液，连续培养21d。取诱导21d细胞，PBS漂洗3min，4%多聚甲醛4℃固定30min。漂洗后用1%阿利新蓝冰醋酸溶液（PH2.5）室温染色30min，蒸馏水漂洗2min，0.1%中性核固红溶液染色5min。蒸馏水冲洗后置于倒置显微镜系统成像。