小鼠实验取材要点

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1、3TissueHarvestProtocolSet-Up:Ifplasmaisdesireduse1ccsyringesw/25Gneedlecoatedw/heparin(withdraw1000U/mlheparindirectlyintosyringeandpullplungerbackandforthtocoatinsideofsyringe.Expungealltheheparinfromthesyringeandtheneedletip)REFERtoEXPERIMENTFORMFOREXPLICITDIRECTIONSastoWHATKINDofhepa

2、rinisneeded1metaltraylargeenoughtofitmousew/boardinbluepadssacpackincluding:1pairsofhemostats1scissor1forcep2-4microclamps(sizedoesn’tmatter)coldPBS(useabout50ml/animal)2%paraformaldehyde(useabout50ml/animal)2-250mllactatedringerssolutionsbagsempty.Cutslitacrossonesideattopofbaginordert

3、ofillwithsolution.Attachfittedtubingonendofbag(sametubingashepatocytetubing).Attachthreewaystopcocktoendoftubing.Attach18Gneedletothestopcock.Fillonebagw/coldPBS(dependinguponhowmanymiceyouhavetosac)Fillsecondbagw/2%para(dependinguponhowmanymiceyouhavetosac)Procedure:Hangbagsonhooksprov

4、idedinthehood.**Bagshouldbe~1meter(3ft)abovesurfaceleveltogetappropriateflowandpressureforperfusion.Shaveabdominalareaandremoveexcesshairwith4x4gauzepad.Midlineincisionthroughskinandabdomenallthewayuptothexyphoid(makesuretopickskinupwhencuttingtoavoidorgandamage)Cutdiaphragmalongribcage

5、toseparateitCutribcagejustlateraltotheR&Linternalthoracicarteries.Placehemostatsoncutxyphoidsegmentwithcurvatureofhemostatfaceuptoholdforviewingheartforcardiacpuncture.Performcardiacpuncture:Use25G5/8needlewith1mlsyringeforcardiacpuncture(mice)Use18Gneedlew/5mlsyringeforcardiacpuncture(

6、rats)InsertneedleintoRventricleofheartandremoveblood*Allowbloodflowtocontrolsuctionofsyringe.PullingtoofastwillcausecellstolysePutbloodintoeppendorfwith5ulofheparin1000Units/ml.(ifcollectingplasmanoheparinforserum)*seebelowstepsforindividualbloodcollectioncircumstancesTissueProcessingan

7、dHarvesting3Aftercardiacpunctureremoveotherorgansasgentlyandpromptlyaspossible.ForSerum:NoHeparinduringcollectionatallUsea25Gneedlew/1mlsyringeforbloodwithdrawalinmice(18Gw/5mlsyringeforrats)*takeofftheneedlebeforeputtingbloodintoeppendorftopreventlysingcellsLetsitin4°overnight