3HThymidine Uptake by Cultured Cells 细胞毒理实验 资讯...
来自 : 蚂蚁淘
Materials
FibroblastcellsinlogphasegrowthCa
,Mgfree-phosphatebufferedsaline(PBSA)5%(w/v)Glutaraldehyde(GTA)2%(w/v)PerchloricAcid(PCA)Subbedslides(coatedwithchromalumgelatin)andPermountNuclearTrackEmulsion(KodakorIlford)Darkroomandchemicalsforphotographicprocessing- Dektoldeveloper
- KodakFixer
Giemsastain,gradedseriesofalcohols,xylolProcedure
- GroweitherLcells(Mousefibroblasts)orchickembryofibroblastsoncoverglassesandthengivethem
H-thymidineforashortperiodoftime. - Attheendofthelabelingperiod,washthecoverslipsinPBSAbygentlygraspingacoverslipwithforcepsandpassingitthroughabeakerofsaline.
- Fixculturesinglutaraldehydefor15minutes.
- Washinseveralchangesofwater.
- Washincold2%PCAfor5minutestoremoveunincorporatedlabeledprecursorstoDNA.Repeattwice.
- Washinwater5minutes.Repeat.
- Drythebacksofthecoverslipswithfilterpaper,andmountCELLSIDEUPonslideswithPermount.Theslidesshouldbeveryclean.Coattheslidesbeforehandwithchromalumgelatin(CAG)bydippingtheslidesintoCAGsolutionanddraininguntildry.Thiscoating,andthegelatincoatingonthecoverslips,helptopreventtheemulsionlayer(below)frompullingawayfromtheslideduringlaterdevelopment.
- AllowPermounttodryovernight.
- Inadarkroom,spoonoutasmallamountofgelintoasuitablevessel,andslowlymeltitat45°Cinawaterbath.KodakNTB-3emulsionisstoredrefrigeratedasagelinascrewcapbottleinsideadoublelight-tightbox.
- Diptheslideintheemulsionanddrainmomentarily.Placetheslidesverticallyonatesttuberackinanovensetat28°Cforatleast1hourindarkness.Ingeneral,theslidesshouldbedriedatatemperaturegreaterthanwillbeusedfordeveloping.Thisminimizesundesirableseparationofemulsionfromtheslide.
- Placetheslidesinlight-tightboxescontainingdessicantandstoreat4°C.
- DevelopasampleslideinDektoldiluted1partdeveloperto2partsdistilledwaterat18°Cfor90seconds.Thetemperatureofthedeveloperwillcontrolthesizeofthesilvergrains.Increasedlengthincreasesbackgroundfogofdevelopment.
DeveloptheslidesasindicatedinChapterTwo.Thelengthofexposure(refrigeratorstorage)mustbedeterminedforeachsystem(afunctionofspecificactivityoflabelinmedium,poolsizes,lengthoflabeling,syntheticrate,etc.).Thusextracontrolslidesarealwaysincludedtoallowrepeatedsampledevelopinguntilausefulnumberofsilvergrainshaveaccumulated.
- Passtheslidesthroughtwochangesofdistilledwater,andintophotographicfixerat18°C.Fixfor5minutes.
- Washslidesintwochangesofdistilledwater,foratotalof5minutes.
- StainthecellswithGiemsadiluted1:30asrequired,ordryslidesslowlyinadust-freeatmosphere,andstainlater.
- Washoffexcessstainbrieflyindistilledwater,dipslidebrieflyin70%ethanol,dehydratein95%and100%alcohol,andclearinxylene.MountcoverslipwithPermount.
- Examinetheslideswithamicroscopeat10Xmagnificationandlookforclustersofsilvergrainsoverthecells.Countandcalculatethepercentofcellsthatarelabeled.
- Examinetheslidesat40Xmagnification.Countthenumberofgrainspercell.
- Prepareahistogrambyplottingthenumberofcellsversusthenumberofsilvergrains.
免责声明 本文仅代表作者个人观点，与本网无关。其创作性以及文中陈述文字和内容未经本站证实，对本文以及其中全部或者部分内容、文字的真实性、完整性、及时性本站不做任何保证或承诺，请读者仅作参考，并请自行核实相关内容。
版权声明 未经蚂蚁淘授权不得转载、摘编或利用其他方式使用上述作品。已经经本网授权使用作品的，应该授权范围内使用，并注明“来源：蚂蚁淘”。违反上述声明者，本网将追究其相关法律责任。
