苏州蚂蚁淘生物科技有限公司是Emfretanalytics血管抗体代理商

EMFRET Analytics—专注脉管系统抗体

德国EMFRET公司专注心血管和血液系统研究用抗体研发和生产，供应的抗体包括清除血液中血小板、流式细胞术鉴定分析小鼠血小板表面糖蛋白，EMFRET公司的抗体不仅适用于流失分析、WB检测，还可用于免疫组化/免疫荧光及免疫共沉淀检测。

EMFRET公司的抗体产品目录：

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TechnicalProtocols

 Flowcytometricanalysisofmouseplateletsurfaceglycoproteins

        - Preparationofdilutedorwashedwholeblood  

      - Preparationofwashedmouseplatelets  

         - Single-coloranalysisofplateletsurfaceglycoproteinexpression 

        - Two-coloranalysisofplateletactivation

 Immunohistochemistry(withacetone-fixedfrozensections)

 Immunoprecipitation

 Immunoblot(WesternBlotAnalysis)

Flowcytometricanalysisofmouseplateletsurfaceglycoproteins

Buffersandreagents

Trisbufferedsaline/Heparin(TBS/Hep):20mMTris/HCl;137mMNaCl;pH7.3;containing20U/mlHeparin.

Phosphatebufferedsaline(PBS):137mMNaCl;2.7mMKCl;1.5mMKH2PO4;8mMNa2HPO4;pH7.14.

Tyrode’sBuffer(modified):134mMNaCl;0.34mMNa2HPO4;2.9mMKCl;12mMNaHCO3;20mMHepes;pH7.0;5mMglucose;0.35%(w/v)bovineserumalbumin

Apyrase:10U/mlstockinH2Obidest(storedat-20°C)

Prostacyclin(Prostaglandin2,PGI2):1mMstockinH2Obidest(storedat-20°C)

CaCl2:1MstockinH2Obidest

Preparationofdilutedorwashedwholeblood

1. Take50µlbloodwithaheparinizedglasscapillary(e.g.ringcap)fromtheretro-orbitalplexusinto1.5mltubescontaining200µlofTBS/Heparin(20U/ml).

2. Dilutein1mlofTyrode´sbufferanduseforflowcytometricanalysis. 

3. Topreparewashedblood,centrifugethedilutedbloodat900xgfor5min,removethesupernatantandresUSPendthepelletin1.25mlTyrode´sbuffer.

4. Add1mMCaCl2directlybeforethestartoftheexperiment.
   

Note:Forthrombin-inducedplateletactivation,plasmahastoberemovedtopreventanti-thrombinactivity

Preparationofwashedmouseplatelets

Thepreparationofwashedplateletsisamoretimeconsumingmethod,thatrequiresalargeramountofblood,butyieldsaplateletpreparationthatcanbeusedforseveralhours.

1. Collect0.5-1mlbloodwith1.5cmglasscapillariesfromtheretro-orbitalplexusintoa1.5mltubecontaining200µlofTBS/Heparin(20U/ml).

2. Centrifugethesamplefor5minat500xgandtransfertheplateletrichplasma(PRP)intoanewtube.Forbestrecoveryofplatelets,takethecompletewhitephaseincludingsomeredbloodcells.

3. Centrifugetheplateletsuspensionfor8minat300xg,andtransferthePRPwithoutanyredbloodcellsintoanewtube.

4. Add0.5µMprostacyclin(PGI2)andcentrifugeat1300xgfor5min.

5. Resuspendtheplateletpelletin1mlTyrode’sbuffer,add0.02U/mlapyraseand0.5µMprostacyclin,incubatefor5minat37°C,andcentrifugefor5minat1300xg.

6. Repeatstep5andresuspendtheplateletpelletin0.5mlTyrode’sbuffer,add0.02U/mlofapyraseandincubatefor30minat37°C.

Singlecoloranalysisofplateletsurfaceglycoproteins

1. Combine5µlofspecificornegativecontrolantibodiesand25µldilutedwholeorwashedbloodintheassaytubeandvortexmixfor1-2seconds.

2. Incubatefor15minatroomtemperature.

3. Stopreactionbyadditionof400µlPBSandanalyzewithin30min.

SampleswereincubatedwithFITC-conjugatedcontrolIgG(blackline),anti-GPVI,oranti-GPVasdescribed.Plateletsweregatedbyforward(FSC)/sidescatter(SSC)characteristics.Fluorescence1(FL1)intensityofthegated(R1)plateletsisshowninseparatehistograms.RBC:redbloodcells.

Twocoloranalysisofplateletactivation

1. Add5µlspecificornegativecontrolantibodiestotheassaytubetogetherwith5µlofapan-plateletMarker.

2. Atthispoint,anyadditionalreagents(e.g.inhibitors)areaddedinsmallvolumes(<5µl).

3. Add26µldilutedwholeorwashedbloodorwashedplatelets(1million)andvortexmixfor1-2seconds.

4. Incubatefor15minatroomtemperature.Stopreactionbyadditionof400µlPBSandanalyzewithin30min.

WashedmouseplateletswereincubatedwithPBS(resting)orthrombin(0.1U/ml)incombinationwithbothFITC-andPE-conjugatedmAbs.PlateletsweregatedbyexpressionofGPIXorGPIIbIIIa(FL1;gate1).

Immunohistochemistry(withacetone-fixedfrozensections)

Pleasenote:Itisnotrecommendedtoperformimmunestainingon(para-)formaldehyde-fixedsamples,sincemostratantibodiesyieldonlypoorresultsundertheseconditionsonmousetissues.
 

1. Mountsectionsfromfrozentissueonslidesandfixinicecold100%acetonefor20minat4°C.DrysamplesovernightatRT.

2. Tominimizereagentvolumes,ahydrophobicringmaybedrawnwithawaterrepellentpenaroundthetissuesectionontheslide.Duringallfollowingprocedures,thisencircledareashouldnotdryup.Therefore,itisrecommendedtoperformincubationstepsinahumidbox.

3. IncubateslidesinPBSfor20min.

4. Whenusingperoxidase-conjugatedsecondaryantibodies,incubateslideswith0.03%H2O2for10minatRTtoinhibitendogenousperoxidaseactivity.RinseslidesthreetimesinPBSfor5mineachwash.

5. Incubateslidesfor1houratRTinblockingsolution(serumfromhostspeciesofsecondaryantibodytobeused),diluted1:10or1:100inPBS.

6. Coverslideswithprimaryantibody(2-10µg/ml)inblockingsolutionandincubatefor2hatRT.

7. BlotexcessliquidfromslidesandrinsethreetimesinPBSfor5mineachwash.

8. Coverslideswithfluorophore-orperoxidase-conjugatedsecondaryantibody(e.g.affinitypurifieddonkeyanti-ratIgG-FITCor-HRP,JacksonImmunoResearch,WestGrove,PA)dilutedinblockingsolutionaccordingtothemanufacturer’sinstructions.Incubatefor1hatRT.

9. BlotexcessliquidfromslidesandrinsethreetimesinPBSfor5mineachwash.

10. a)Fluorescentlystainedsamplescanbedirectlyanalyzedbyfluorescencemicroscopy.

11. b)Forvisualizationofperoxidase-labeledstructures,coverslideswithfreshlyprepared3-amino-9-ethylcarbazole(AEC)substrateandincubateuntilredstainingisvisIBLeunderthemicroscope,timecanvarybetween5and30min.Stopreactionbyrinsingwithdeionizedwaterbeforeorangebackgroundstainingoccurs.Forcounterstaining,incubatesamplesfor30-240swithhematoxylinandrinseslidesfor20minwithrunningwarmwater.Mountsectionswithaqueousembeddingsolution(e.g.Aquatex,Merck).

Immunoprecipitation

BuffersandReagents

PBS/EDTA:137mMNaCl,2.7mMKCl,1.5mMKH2PO4,8.0mMNa2HPO4x2H2O,pH7.3;5mMEDTA

IPbuffer:40mMTris/HCl,0.3MNaCl,1mMEDTA,2mMPMSF,10µg/mlLeupeptin,10µg/mlAprotinin,1µg/mlPepstatin

Igepal:10%stockinH2Obidest

2xSDS-samplebuffer:10%beta-mercaptoethanol(forreducingbuffer),10%Trisbuffer(1.25M),20%Glycerin,4%SDS,0.02%Bromophenolblue 

1. Washmouseplateletsorcells(10millionperimmunoprecipitation)3xin1mlPBS/EDTA(5min,1300xg)andresuspendinIP-buffer.

2. Lyseplateletsbyadditionof1%Igepalfor15–20minatRT.

3. Toremovecelldebris,spin5minat10,000xgandtransfersupernatanttoanewcup.

4. Preclearbyadding20µlofwashedproteinGagarose(PGA).Incubatewithrotationforatleast3hat4°C.

5. Centrifugethesamplesfor5minat3000xg.Fillsupernatantsintonewtubes,addtheantibodyataconcentrationgiveninthedatasheet(usuallybetween2and5µg/ml),andafter30minadd25µlofwashedPGA.Incubatewithrotationforatleast2hat4°C,preferablyovernight.

6. Towashtheprecipitate,centrifugethesamplesfor1minat3000xg.Add1mlof1xIPbuffercontaining1%NP-40tothePGApellet.Centrifugefor1minat3000xg.

7. WashthePGApellettwiceinplainIP-buffer(1minat3000xg).

8. ResuspendthePGApelletin25µlof1xSDS-samplebuffer(reducingornon-reducing)andboilfor5min.Thesamplescanthenbefrozen(-20°C)forlateruse,orSDS-PAGEandsubsequentimmunoblotanalysiscanbeperformed.

Immunoblot(WesternBlotAnalysis)

Buffersandreagents

Lysisbuffer:40mMTris/HCl,0.3MNaCl,1mMEDTA,0.05%NaN3,1%NP-40

PBS:137mMNaCl,2.7mMKCl,1.5mMKH2PO4,8.0mMNa2HPO4x2H2O,pH7.3

PBS-5%milk:PBScontaining5%non-fatdrymilk

PBS/T:PBScontaining0.1%Tween20

PBS/T-1%milk:PBS/Tcontaining1%non-fatdrymilk

Allstepscanbeperformedatroomtemperature:

1.   Preparelysateofmouseplateletsorcellsinlysisbufferandincubatewith(2x)SDSsamplebufferfor5minat95°C.

2.   SeparateproteinsbySDS-polyacrylamidegelelectrophoresis(SDS-PAGE)underreducingornonreducingconditions,asindicatedintherespectivedatasheet,andtransfertheproteinstoPVDFmembrane.

3.   BlockthemembranewithPBS-5%milkfor1hwithagitation.

4.   Incubatethemembranewith2-5µg/mlofprimaryantibodyinPBS/T-1%milkfor1hwithagitation.

5.   RinsethemembranetwicewithPBS/Tfollowedbytwo30minwashingperiodsinPBS/T.Washingproceduremaybereducedtothreeperiodsof10mineach,butthatshouldbedeterminedindividually.

6.   Incubatewithsecondaryantibody(HRP-conjugatedanti-ratIg,e.g.fromDAKO,Glostrup,Denmark,#P0162)inPBS/T-1%milk

for45–60minwithagitation.

7.   RinsethemembranetwicewithPBS/Tfollowedbytwo30minwashingperiodsinPBS/T.

8.   VisualizeboundantibodywithECL(enhancedchemiluminiscence).