Description
Mannosereceptortargetingbymannosylatedliposomeshasbeendemonstratedforavarietyofmannosylatedlipidconjugatesinavarietyofliposomemorphologiesandcompositionsinseveraldifferent invitro and invivo models.Averylargenumberofpublicationsisaboutusingahydrophobicderivativeofmannose(4-aminophenylalpha-D-mannopyranoside)ratherthanusingamannosylatedlipidinClodronateliposomes.Thisismainlyduetothehighcostandcomplexityofsynthesizingandconjugatingmannosetolipid.4-aminophenylalpha-D-mannopyranosideiscommerciallyavailableandfarlessexpensivethansynthesizingmannoseconjugatedlipid.
Whymannose?Mannoseisoneofthecarbohydratecomponentsofmanybacterialandviralcellsurfaces;therefore,theever-efficient,highlyredundantimmunesystemhasevolvedmultiplemechanismsforidentifyingpathogensbasedonmannoserecognition.Theanimalandplantkingdomslikewiseutilizecarbohydraterecognitionsignalingmechanismsincludingmannoseresidues.Manypublicationsevaluateothercarbohydratesastargetingmechanismsforvariouscelltypes,howevermannosetargetingtophagocytesappearstobeoneofthemorespecificmechanismsidentifiedtodate.Mammaliancellsurfaceidentificationmoleculesbasedonmannosebinding,suchastheICAMfamilyofleukocyteadhesionmolecules,targettheSIGNfamilyofmannosereceptorstoaccomplishself-recognition invivo.
Awell-knownandcitedstudybyUmezawa&Eto [1]demonstratesthatliposomescontainingaminophenylmannosideweremostefficientlyincorporatedintothemousebrainacrossthebloodbrainbarrier.TherADIolabeledliposomesbearingaminophenylalpha-D-mannopyranosideweremaximallyincorporatedintothemousebrainafter48hours,whereasinthespleenandliver,theseradioactivitiesweremaximumafter12hours.Thestudiesalsoshowedthatliposomesweremostincorporatedwasglialcellsratherthanneuronalcell.Thesubcellularfractionationstudyindicatesthatmannoselabeledliposomesareincorporatedintolysosomesrichfractionbothinliverandbrain.
Therearefivemannosylatedfluorescentcontrolliposomeproducts(m-Fluoroliposome®)form-Clodrosome®(mannosylatedclodronateliposomes).Allfivemannosylatedfluorescentliposomesincorporatealipophilicdyeinsidetheirmembranes.Theyareinsolubleinwater;however,theirfluorescenceiseasilydetectedwhenincorporatedintomembranes.DiI,DiO,DiD,DiRandDiAcoverawiderangeofexcitationandemissionwavelengthsfrom300sto900s.DiIandDiOhavefluorescenceexcitationandemissionmaximaseparatedbyabout65nm,facilitatingtwo-colorlabeling.TheemissionspectrumofDiAisverybroad,allowingittobedetectedasgreen,orange,orevenredfluorescencedependingontheopticalfilterused.DiI,DiO,DiDandDiRbelongtothedialkylcarbocyaninesfamilyofcompounds.Thespectralpropertiesofthedialkylcarbocyaninesarelargelyindependentofthelengthsofthealkylchainsbutareinsteaddeterminedbytheheteroatomsintheterminalringsystemsandthelengthoftheconnectingbridge.Theyhaveextremelyhighextinctioncoefficients,moderatefluorescencequantumyields,andshortexcitedstatelifetimesinlipidenvironments(~1ns).Thefluorescencespectrumofeachdyeisshownbelow.
Youcanchoosethem-Fluoroliposome®basedonthetypeofthefluorescentequipmentandfiltersthatyouuseinyourlab.Mannosylatedclodronateliposomescannotbemadefluorescentsimplyduetothepotentialforinaccurateand/oruninterpretabledatabeinggeneratedbylabelledm-Clodrosome®.Formoreinformation,pleaserefertothetechnicalnotesection.
FormulationInformation
Clodrosome®LiposomalClodronateSUSPension
| LipidComposition | Concentration(mg/ml) | Concentration(mM) | MolarRatioPercentage |
|---|---|---|---|
| Total | 23mg/ml | 35.1mM | 100 |
| L-alpha-Phosphatidylcholine | 18.8 | 24.3 | 70 |
| Cholesterol | 4.2 | 10.9 | 30 |
| EncapsulatedDrug | Concentration |
|---|---|
| Clodronate((Dichloro-phosphono-methyl)phosphonate),DisodiumSalt | 18.4*mM |
| *Dependingonthetypeoftheclodronatesalt,itsconcentration(mg/ml)varies.Iftetrahydratesaltisused,theconcentrationoftheencapsulateddrugwillbe~7mg/ml,andifanon-hydratedsaltisused,theconcentrationwillbe~5mg/ml. | |
Encapsome®ControlLiposomeSuspension
| LipidComposition | Concentration(mg/ml) | Concentration(mM) | MolarRatioPercentage |
|---|---|---|---|
| Total | 23mg/ml | 35.1mM | 100 |
| L-alpha-Phosphatidylcholine | 18.8 | 24.3 | 70 |
| Cholesterol | 4.2 | 10.9 | 30 |
Fluoroliposome®-DiA
| LipidComposition | Concentration(mg/ml) | Concentration(mM) | MolarRatioPercentage |
|---|---|---|---|
| Total | 23mg/ml | 35.1mM | 100 |
| L-alpha-Phosphatidylcholine | 18.8 | 24.3 | 70 |
| Cholesterol | 4.2 | 10.9 | 30 |
| Mannosylation | Concentration |
|---|---|
| 4-Aminophenyl-alpha-D-mannopyranoside | 9.53mol% |
| FluorescentDye | Excitation/Emission(nm) | Concentration(mg/ml) | Concentration(mM) |
|---|---|---|---|
4-(4-(Dihexadecylamino)styryl)-N-methylpyridiniumIodide(DiA) | 456/590 | 0.0625 | 0.0794 |
| BufferandLiposomeSize | Specification |
|---|---|
| Buffer | PhosphateBufferedSaline |
| pH | 7.4 |
| LiposomeSize | 1.5-2µm |
TechnicalNotes
- Toreachbloodstream-accessIBLe,mannose-receptorpositivecellsoutsidetheliver,asignificantnumberofliposomeswillhavetoescapefirst-passuptakebytheliverandspleen,sothatthetargetcellsareexposedtoahigherconcentrationofmannosylatedliposomesfromtheblood.Onestrategythathasbeenusedtoensurethatliposomesescapetheliverandspleenisknownasreticuloendothelialsystem(RES)blockadeinwhichanimalsarepre-dosedwithasufficientquantityofliposomestotemporarilysaturatethephagocyticcellsoftheblood,liverandspleen,alsoknownasthereticuloendothelialsystem(RES)orthemononuclearphagocytesystem(MPS).Thissufficientquantityisdependentupontheliposometypeandcompositionaswellasthespeciesbeingdosed;thepre-dosedliposomesdonotnecessarilyneedtobethesametypeorcompositionasthetherapeuticordiagnosticliposomesavoidingtheRES.Soonafterthispre-doseisclearedfromthebloodstream(usuallywithinacoupleofhours),theliposomesofinterestaredosed.SincetheRESisinvolvedindigestingthepreviousdoseofliposomes,thesubsequentlydosedliposomeswillremaininthecirculationmuchlongerthusbemuchmorelikelytobindtotheirtargetsiteoutsidetheRESincludingthosephagocyticcellswhichareaccessible,butarenotusuallyexposedtoahigherconcentrationofliposomes.
- WhileRESblockadeisusuallythoughtofassaturatingphagocyticcells,ithasbeenshownthatopsonin-bindingbyliposomesisasaturablephenomenon.Therefore,partofRESblockademayinvolveserumdepletionofcomplementandotheropsoninsknowntocoatliposomes.Inthecurrentapplication,removalorreductionintheconcentrationofsolublemannose-receptorsmayfurtherincreasetheprobABIlityofamannosylatedliposomebeingabletointeractwithmannosereceptorsonthetargetcell.Therefore,ifthegoalistodepleteatargetsubsetofmannose-receptor+cellswhichmaynotnormallybeexposedtoasubstantialnumberofmannosylatedliposomes,pre-dosingwithmannosylatedclodronateliposomes,inordertobothsaturatetheblood,liverandspleenphagocytesandreducetheconcentrationofopsoninsincludingsolublemannosereceptors,shouldincreasethenumberofsubsequentlydosedmannosylatedclodronateliposomesavailabletothistargetsubsethypotheticallyresultinginincreaseduptakeanddepletionbythesetargetedcells.
- TheissuewithfluorescentClodrosome®hastodowiththepotentialforinaccurateand/oruninterpretabledatabeinggeneratedbylabelledClodrosome®.WhenClodrosome®inducesmacrophageapoptosis,thefluorescentlipidincorporatedintotheClodrosome®thatisdisruptedandmetabolizedinthephagolysosomewillbedispersedamongtheresidualapoptoticbodieswhicharesubsequentlyphagocytosedbyothermacrophages.Therefore,fluorescentlipidmaybedetectedinphagocyticcellswhichneverphagocytosedClodrosome®especiallywhenFACSorfluoroscopyareutilizedtodetectfluorescentcells(FACS)orfluorescencelevelsinatissuehomogenate(fluoroscopy).Anotherpotentialartifactarisesfromfluorescentlipidremainingintheextracellular“garbage”,whichhasnotyetbeenclearedbyotherphagocytes,generatingahighbackgroundfluorescence.However,experiencedconfocalmicroscopistmaybeabletodifferentiatebetweenthepunctatefluorescenceresultingfromfluorescentintactliposomesversusthemorediffusefluorescencecharacteristicofdisruptedliposomesandsomehavesuccessfullyusedfluorescentclodronateliposomestovisualizethecellularlocationoftheseliposomesbyconfocalmicroscopyinvivo[2].Afurthercomplicatingfactoristhatpublisheddatavarieswidelyastoexactlywhenclodronateliposomesbegintoinduceapoptosisinmacrophages.Mönkönnnen etal.showthatmacrophagedeathismeasurablewithinthefirsthourafterclodronateliposometreatmentonRAW264cellsinvitro[3],whileothershavereportednosignsofmacrophageapoptosisuntilseveralhoursaftertreatmentinvivo.Thevariabilityinthedataislikelyduetodifferentliposomalformulationsofclodronateaswellasthevastlydifferentexperimentalconditions.Therefore,aswithmostBIOLOGicalstudies,especiallythoseinvolvingliposomes,theamountoftimebetweentreatingtheanimalorcellswithclodronateliposomesandtheonsetofapoptosiswillneedtobeestablishedineachexperimentalmodel.IfthenatureoftheresearchdemandsthatClodrosome®betrackedratherthanthecontrol,EncapsulacanprovideDiI-labelledClodrosome®uponrequest,andassumingthattheClodrosome®distributioncandefinitivelybeassessedpriortotheonsetofapoptosis,clearandvaliddataonthebiodistributionoffluorescentClodrosome®shouldbeobtainable.Still,formostpurposes,Fluoroliposome®(fluorescentcontrolliposomes)willprovidetherequireddatawithfarfewerpotentialartifacts.
- Whenmonitoringmonocyteuptakeinvivoinnormalanimals,thecirculatingmonocytesmay“disappear”orshowreducedcountswithinthefirst2hpost-injectionduetomarginationofthemonocytespost-liposomephagocytosis.Thesecellswillre-enterthecirculationwithinafewhours.Sunderkötter etal.demonstratethisphenomenonanddiscussthebehaviorindetail.Alsoconsiderthatcirculatingmonocyteshavealifetimeofabout24hsolabeledmonocyteswillbecontinuallyleavingthecirculation,eveninnormalanimals,duetoagingofthemonocytes[3].
- WhenanimalsorcellsaretreatedwithClodrosome®,phagocyticcellsrecognizetheliposomesasinvadingforeignparticlesandproceedtoremovetheliposomesfromthelocaltissueorserumviaphagocytosis.Theliposomesthenreleaseclodronateintothecytosolresultingincelldeath.Unencapsulatedclodronatecannotcrossthecellmembranetoinitiatecelldeath.
- Encapsome®controlliposomesarerecognizedandphagocytosedbythesamemechanismasClodrosome®.Sincethecontrolliposomesdonotcontainclodronate,thephagocyticcellsarenotkilled.However,phagocytesdorespondtotheingestionofthecontrolliposomesbycytokinesecretion,temporarysuspensionofphagocyticactivityandotherresponsesdescribedintheliterature.
- Theproductmustberemovedfromthevialusingsteriletechnique.Donotuseifsterilityiscompromised.Thisisparticularlyimportantifasinglevialisaccessedmultipletimesoverseveralweeks.Theproductshouldnotbeusedmorethan60daysafterreceipt,evenifunopened.
- Liposomesmaysettlewhenleftundisturbedformorethanafewhours.Immediatelypriortouse,inordertoensureahomogeneousliposomesuspension,slowlyinvertthevialseveraltimesuntilthesuspensionappearshomogeneousbyvisualinspection.Vigorousorerraticshakingwillnotdamagetheliposomesbutmayinducefoamingandbubbleformationmakingitmoredifficulttoaccuratelymeasurethedesireddosage.
- Ifthepersonnelperformingintravenousinjectionsarenotexperiencedinorfamiliarwith,precautionsforinjectinglargervolumes(~10%animalweightinml),viscousliquidsorparticulatesuspensions,considerhavingextraanimalsavailableincaseseriousinjection-relatedadverseeventsoccur.Dosecontrolanimalsfirsttobecomefamiliarwithlargevolumeinjections.
- WithinhoursaftersystemicadmiNISTrationofClodrosome®,animalsbegintoloseimportantcomponentsoftheirimmunesystem.Standardanimalhandlingandhousingprotocolsarenotsuitableforimmunocompromisedanimals.Evenwhensuchprecautionsaretaken,monitorthegeneralhealthofeachanimalforopportunisticinfectionsunrelatedtotheexperimentalprotocol.Thereisnoinherenttoxicitytotheproductattherecommendeddoselevels.
- Whendosingintravenously,usestandardprecautionsfordosinglargervolumestoanimalsincludingthefollowing:a)warmproducttoroomtemperaturepriortodosing;b)ensurethatallairbubblesareremovedfromthesyringepriortodosing.Intravenousinjectionofairbubblesmayresultinairemboliwhichcankillorseriouslyinjureanimals;c)injectproductataslow,steadyrateofnomorethan1ml/min;d)decreaseinfusionrateifanimalsdisplayanyatypicalreactionssuchasunusualagitation.
- Infusion-relatedadversereactionsusuallyinvolvetheanimalgaspingforairorotherseizure-likemovements.Animalsoftenrecoverwithnoapparentpermanentinjury,butanypotentialeffectsonexperimentalresultsmustbeassessedbytheresearcher.
- Liposomesshouldbekeptat4°CandNEVERbefrozen.
Dosage
Appearance
m-Clodrosome®andm-Encapsome®arebothwhitemilkysuspensions,andm-Fluoroliposome®-DiAisayellowliquidsuspension,allmadeoflargemicrosizemultilamellarliposomes.Duetotheirlargesize,someliposomesmightsettletothebottomofthevial.Ifleftsittingidleintherefrigerator,m-Encapsome®andm-Fluoroliposome®-DiAwillphaseseparateandformpelletsinthebottomofthevialleavingaclearsolutionontop.m-Clodrosome®mightdothesameonlynotasseverely.Therefore,bothshouldbegentlyshakennottoformbubblesbuttoformahomogeneoussolutionpriortouse.
EducationalVideos
Ordering/ShippingInformation
- Allliposomebasedformulationsareshippedonblueiceat4°Cininsulatedpackagesusingovernightshippingorinternationalexpressshipping.
- LiposomesshouldNEVERbefrozen.Icecrystalsthatforminthelipidmembranecanrupturethemembrane,changethesizeoftheliposomesandcausetheencapsulateddrugtoleakout.Liposomesinliquidformshouldalwaysbekeptintherefrigerator.
- ClientswhoorderfromoutsideoftheUnitedStatesofAmericaareresponsiblefortheirgovernmentimporttaxesandcustomspaperwork.EncapsulaNanoSciencesisNOTresponsibleforimportationfeestocountriesoutsideoftheUnitedStatesofAmerica.
- WestronglyencouragetheclientsinJapan,Korea,TaiwanandChinatoorderviaadistributor.Toughcustomsclearanceregulationsinthesecountrieswillcausedelayincustomclearanceoftheseperishableformulationsifordereddirectlythroughus.Distributorscaneasilyclearthepackagesfromcustoms.Toseethelistofthedistributorsclickhere.
- ClientsorderingfromuniversitiesandresearchinstitutesinAustraliashouldkeepinmindthattheliposomeformulationsaremadefromsyntheticmaterialandtheformulationsdonotrequirea“permittoimportquarantinematerial”.LiposomesareNOTbiologicalproducts.
- Ifyouwouldlikeyourinstitute’sFedExorDHLaccounttobechargedforshipping,thenpleaseprovidetheaccountnumberatthetimeofordering.
- EncapsulaNanoScienceshasnocontroloverdelaysduetoinclementweatherorcustomsclearancedelays.YouwillreceiveaFedExorDHLtrackingnumberonceyourorderisconfirmed.ContactFedExorDHLinadvanceandmakesurethatthepaperworkforcustomsisdoneontime. AllsubsequentshippinginquiriesshouldbedirectedtoFederalExpressorDHL.
StorageandShelfLife
Storage
m-Clodrosome®,m-Encapsome®andm-Fluoroliposome®shouldalwaysbestoredatinthedarkat4°C,exceptwhenbroughttoroomtemperatureforbriefperiodspriortoanimaldosing.DONOTFREEZE.Ifthesuspensionisfrozen,clodronatecanbereleasedfromtheliposomesthuslimitingitseffectivenessindepletingmacrophages.ENSisnotresponsibleforresultsgeneratedbyfrozenproduct.
ShelfLife
m-Clodrosome®,m-Encapsome®andm-Fluoroliposome®aremadeondailybasis.Thebatchthatisshippedismanufacturedonthesameday.Itisadvisedtousetheproductswithin60daysofthemanufacturingdate.
Referencesandbackgroundreading
1.UmezawaFA,EtoY.Liposometargetingtomousebrain:mannoseasarecognitionMarker.Biochemicalandbiophysicalresearchcommunications.1988Jun30;153(3):1038-44.
2.PolflietMM,GoedePH,vanKesteren-HendrikxEM,vanRooijenN,DijkstraCD,vandenBergTK.Amethodfortheselectivedepletionofperivascularandmeningealmacrophagesinthecentralnervoussystem.J.Neuroimmunol.2001Jun1;116(2):188–95.
3.MönkkönenJ,LiukkonenJ,TaskinenM,HeathTD,UrttiA.Studiesonliposomeformulationsforintra-articulardeliveryofclodronate.JournalofControlledRelease.1995Aug;35(2–3):145–54.
4.SunderkötterC,NikolicT,DillonMJ,vanRooijenN,StehlingM,DrevetsDA,LeenenP.SubpopulationsofMouseBloodMonocytesDifferinMaturationStageandInflammatoryResponse.JImmunol.2004Apr1;172(7):4410–7.
5.NagaiH,KuwahiraI,SchwenkeDO,TsuchimochiH,NaraA,OguraS,SonobeT,InagakiT,FujiiY,YamaguchiR,WingenfeldL.Pulmonarymacrophagesattenuatehypoxicpulmonaryvasoconstrictionviaβ3AR/iNOSpathwayinratsexposedtochronicintermittenthypoxia.PLoSOne.2015Jul1;10(7):e0131923.
6.ZhuY,SoderblomC,KrishnanV,AshbaughJ,BetheaJR,LeeJK.Hematogenousmacrophagedepletionreducesthefibroticscarandincreasesaxonalgrowthafterspinalcordinjury.Neurobiologyofdisease.2015Feb28;74:114-25.
7.YunMH,DavaapilH,BrockesJP.Recurrentturnoverofsenescentcellsduringregenerationofacomplexstructure.Elife.2015;4:e05505.
8.ArwertEN,HarneyAS,EntenbergD,WangY,SahaiE,PollardJW,CondeelisJS.AUnidirectionalTransitionfromMigratorytoPerivascularMacrophageIsRequiredforTumorCellIntravasation.Cellreports.2018May1;23(5):1239-48.
9.ItoT,IshigamiM,MatsushitaY,HirataM,MatsubaraK,IshikawaT,HibiH,UedaM,HirookaY,GotoH,YamamotoA.SecretedEctodomainofSIGLEC-9andMCP-1SynergisticallyImproveAcuteLiverFailureinRatsbyAlteringMacrophagePolarity.Scientificreports.2017Mar8;7:44043.
10.MironVE,BoydA,ZhaoJW,YuenTJ,RuckhJM,ShadrachJL,vanWijngaardenP,WagersAJ,WilliamsA,FranklinRJ.M2microgliaandmacrophagesdriveoligodendrocytedifferentiationduringCNSremyelination.Natureneuroscience.2013Sep;16(9):1211.
11.AndreouK,SarmientoSotoM,AllenD,EconomopoulosV,deBernardiA,LarkinJ,SibsonNR.Anti-InflammatoryMicroglia/MacrophagesasaPotentialTherapeuticTargetinBrainMetastasis.Frontiersinoncology.2017;7:251.
12.AlishekevitzD,Gingis-VelitskiS,Kaidar-PersonO,Gutter-KaponL,SchererSD,RavivZ,MerquiolE,Ben-NunY,MillerV,Rachman-TzemahC,TimanerM.Macrophage-inducedlymphangiogenesisandmetastasisfollowingpaclitaxelchemotherapyisregulatedbyVEGFR3.Cellreports.2016Oct25;17(5):1344-56.
13.OhSH,KimHN,ParkHJ,ShinJY,BaeEJ,SunwooMK,LeeSJ,LeePH.Mesenchymalstemcellsinhibittransmissionofα-synucleinbymodulatingclathrin-mediatedendocytosisinaParkinsonianmodel.Cellreports.2016Feb2;14(4):835-49.
14.KanoF,MatsubaraK,UedaM,HibiH,YamamotoA.SecretedEctodomainofSialicAcid‐BindingIg‐LikeLectin‐9andMonocyteChemoattractantProtein‐1SynergisticallyRegenerateTransectedRatPeripheralNervesbyAlteringMacrophagePolarity.STEMCELLS.2017Mar1;35(3):641-53.
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1）做免疫组化的片子最好不要同时做HE染色，因为那样会掩盖阳性着色的结果；而且用苏木素复染细胞核也是淡染，若要在免疫组化的片子上观察形态学的改变，除非有非常典型的表现，一般是有很大的难度的。
2）你是培养的细胞做免疫组化吗？如果是的话，那就每个样本只有一张片子吧？唯一的办法是进行免疫双染，同时做CD34和AFP（甲胎蛋白）。我觉得后者阳性便可以或多或少的获得向肝细胞分化的证据。
3）用HE染色来识别造血干细胞向肝细胞分化，也就是说要看到肝细胞的典型形态学改变后才可以下结论。问题是：早期的肝细胞仅凭形细胞态学观察并不能很容易的被识别。病理大夫看组织切片，重要的是看其组织学结构，对肝细胞也是这样。如果单拿出来一个细胞，还是培养的细胞，染色后真的不容易区分。所以我觉得你设计的用HE染色来确定早期细胞向哪个方向分化不是很有说服力。
4）如果是切片，也就是说每个样本可以有多张切片，那可以在连续切片上分别染CD34和AFP，拍照时选择相同视野，也有一定说服力。但是还是不如双染法更可靠。
5）用双染最大的问题在于：这两者都表达在胞浆中，染色容易相互覆盖。所以在双染的方法选择上你还要再考虑一下，可不可以用免疫荧光双染？这样也很有说服力的
GlucosestarvationcausestranslocationofAMPKβ2tothelysosomeinHEK-293cellsthatisdependentonN-myristoylation.Theexperimentwasperformedinβ2KOcellsasinFig.1c,exceptthatthelysosomalMarkerLAMP1(taggedwithRFP)wasco-expressedwiththewild-typeormutantAMPKβ2.Upperpanelsshowmergedimagesstainedblue(4′,6-diamidino-2-phenylindole(DAPI),nuclei),red(LAMP1,lysosomes)andgreen(AMPKβ2,detectedusingantibodyvalidatedine),incellsincubatedwithorwithoutglucosefor20 min.Lowersmallpanelsaremagnificationsoftheareasindicatedbydashedboxesintheupperpanels,showing(LtoR)redandgreenchannelsandmergedimages.
下面的这段话是图注，图注的意思我明白，但是我想知道merge后的图看什么颜色的荧光，蓝色是细胞核，红色是lysosome(位于胞质），绿色是AMPKβ2，该实验是想观察AMPKβ2是否转位到lysosome上了，如果确实发生了AMPKβ2转位到lysosome上，那么merge后是红色与绿色融合在一起，是吗？融合在一起发什么颜色的光了？
（2）另一方面，使抗原抗体结合更稳定。一般不需要,但对表达较弱的抗原可能有用,4度和37度时分子运动方式不同,前者分子碰撞机率和运动速度小于后者,后者结合更快,但敏感性也提高了并易造成非特异染色。
（3）其实，我更赞同后一种说法，因为我尝试把肝脏或睾丸片子从4度过夜拿出后，直接用PBS洗没发生过脱片现象。事实胜于雄辩！
有没有朋友使用碧云天的细胞衰老β-半乳糖苷酶染色试剂盒做过细胞衰老，本人小白，求相关细胞处理的过程及数据分析方法。贴壁细胞六孔板需要接种多少，染色后怎样计数进行数据处理。谢谢啦！
石蜡切片组织茜素红钙染色试剂盒产品说明书（中文版）
主要用途
石蜡切片组织茜素红（ALIZARINREDS）钙染色试剂是一种旨在使用标准化的化学分离石蜡方法和鳌和技术，使钙离子和茜素红S产生复合物，来分析存档中的石蜡包埋的组织切片中橘红色钙沉积现象的权威而经典的技术方法。该技术经过精心改良、成功实验证明的。主要适用于石蜡包埋组织切片的钙沉积和钙化结节检测。广泛用于骨细胞或组织病理生理的研究。产品严格无菌，即到即用，操作简捷，性能稳定，显色清晰。
技术背景
茜素红（ALIZARINREDS）是一种蒽醌（anthraquinone）衍生物：9,10-二氢-3,4-二羟基-9,10-二氧代-2-蒽磺酸单钠盐（9,10-Dihydro-3,4-dihydroxy-9,10-dioxo-2-anthracenesulfonicacidsodiumsalt），又称媒介红3（MordantRed3）或茜素磺酸钠（Sodiumalizarinesulfonate）。其分子式为C14H7NaO7S，分子量为342.25。茜素红和钙离子以鳌和方式形成复合物，用以识别组织细胞的钙盐成分。钙盐变化是骨细胞增殖分化和骨组织成骨潜能的标志之一。通过茜素红染色，产生桔红色沉积，但会受到其它金属元素的干扰。
产品内容
脱蜡液（ReagentA）自备
补水液A（ReagentB）100毫升
补水液B（ReagentC）100毫升
补水液C（ReagentD）100毫升
补水液D（ReagentE）100毫升
清理液（ReagentF）30毫升
染色液（ReagentG）10毫升
产品说明书1份
保存方式
保存在4℃冰箱里；染色液（ReagentG），避免光照；试剂具有腐蚀性，注意操作安全。有效保证3月
用户自备
小型染色缸：用于石蜡切片的脱蜡操作
光学显微镜：用于组织细胞染色后观察分析
实验步骤
一、脱蜡处理
1．取出10片待测的石蜡包埋的组织切片（注意：石蜡包埋前，组织切片须用10％甲醛4℃条件下，固定16小时，此步很重要）
2．（选择步骤）放进80℃烘箱，孵育30分钟
3．（选择步骤）室温下静置15分钟
4．按下表依次放进小染色缸里孵育
染色缸
孵育时间
50毫升脱蜡液（ReagentA）
15分钟
50毫升脱蜡液（ReagentA）
15分钟
50毫升脱蜡液（ReagentA）
15分钟
50毫升补水液A（ReagentB）
3分钟
50毫升补水液B（ReagentC）
3分钟
50毫升补水液C（ReagentD）
3分钟
50毫升补水液D（ReagentE）
3分钟
5．小心移去切片上的补水液D（ReagentE）
6．小心加上200微升清理液（ReagentF）在切片上，铺满整个切片样品表面
7．室温下孵育5分钟
8．小心移去切片上的清理液（ReagentF）
二、样本染色处理
1．小心加上200微升染色液（ReagentG），铺满整个切片样品表面
2．室温下孵育2分钟（注意：可以延长至20分钟）；或直至可见桔红色
3．小心移去切片上的染色液（ReagentG）
4．空气中晾干
三、样本澄清处理
1．小心加上200微升补水液B（ReagentC）在切片上，铺满整个切片样品表面
2．小心移去切片上的补水液B（ReagentC）
3．重复实验步骤1和2二次
4．小心加上200微升补水液A（ReagentB）在切片上，铺满整个切片样品表面
5．小心移去切片上的补水液A（ReagentB）
6．重复实验步骤4和5二次
7．小心加上200微升脱蜡液（ReagentA）在切片上，铺满整个切片样品表面
8．小心移去切片上的脱蜡液（ReagentA）
9．重复实验步骤7和8一次
10．放上盖玻片或封片
11．即刻在一般光学显微镜下观察：钙沉积阳性细胞呈现桔红色
注意事项
1．本产品为50次操作
2．操作时，须带手套
3．试剂具有腐蚀性，注意操作安全
4．石蜡包埋前，组织样品须用10％甲醛4℃条件下，固定16小时，否则造成样品支离破碎
5．所有操作在室温下进行
6．试剂溶液在样品表面时，避免有气泡存在，同时确保铺满样品表面
7．样品染色避免过度，肉眼可见桔红色即可终止染色
8．组织细胞染色完成后，即刻进行光学显微镜观察
9．本公司提供系列组织细胞金属元素染色试剂产品
质量标准
1．本产品经鉴定性能稳定
2．本产品经鉴定显色清晰
