ADENOSINE TRIPHOSPHATASE (ATPASE)
来自 : 蚂蚁淘
SECTIONSUnfixedcryostatorcoldFormolcalcuim/gumsucrosefixedonmusclebiopsysections.
SOLUTIONS
a.0.1MGlycinebuffer.
- 0.75gglycine+0.585gNaC1madeupto100mlwithdistilledwater.
b.O.1Mglycine/NaC1bufferwith0.75MCaC12
- 50ml0.1Mglycinebuffer
- 10ml0.75MCaC12-11.03gCaC122.H20/100ml.
- Addapprox.22ml0.1MNaOHuntil9.6-9.8pH
METHODAtpH9.4.
- Incubatefreshlycutsectionsat37Cfor11minutes,inthefollowing
- solution
- 5mgATP.dissolved.Add10mlofsolution(b)adjusttopH9.6-9.8.
- Rinsewellindistilledwater.
- Immersein2%COC12for5minutes.
- Rinsewellindistilledwater+3or4intapwater.
- Immerseindilute(1:10)ammoniumsulphidesolutionfor30seconds.
- Rinsewellinrunningtapwater.
- (optional)staininHarris"shaematoxylin,blueintapwater.
- Mountinglycerinejelly.
METHODAtpH4.2and4.6- Preincubateat4Cin0.1Msodiumacetatebufferwith10mMETDAadded(0.372g/100mlofbuffer)for10minutesatpH4.2or4.6.
- Washindistilledwater.
- ProceedasforthepH9.4method.
NOTES
OnlythepH4.2sectionsneedcounterstaininginhaematoxylin.SectionsmustbeVERYwellwashedaftersteps3and5toremoveanytracesofprevioussolutions
NOTE:NoresponsibilityisassumedbyTheUniversityofNottinghamortheQueensMedicalCentreNHSTrustforanyinjuryand/ordamagetopersonsorpropertyasamatterofproductsliABIlity,negligenceorotherwise,orfromanyuseoroperationofanymethods,products,instructionsorideascontainedinthematerialherein.ItistheusersresponsibiltytoensurethatallproceduresarecarriedoutaccordingtoappropriateHealthandSafetyrequirements.
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