SECTIONS

UnfixedcryostatorcoldFormolcalcuim/gumsucrosefixedonmusclebiopsysections.

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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

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METHODAtpH9.4.

1. Incubatefreshlycutsectionsat37Cfor11minutes,inthefollowing
2. solution
   • 5mgATP.dissolved.Add10mlofsolution(b)adjusttopH9.6-9.8.
3. Rinsewellindistilledwater.
4. Immersein2%COC12for5minutes.
5. Rinsewellindistilledwater+3or4intapwater.
6. Immerseindilute(1:10)ammoniumsulphidesolutionfor30seconds.
7. Rinsewellinrunningtapwater.
8. (optional)staininHarris"shaematoxylin,blueintapwater.
9. Mountinglycerinejelly.

METHODAtpH4.2and4.6

1. Preincubateat4Cin0.1Msodiumacetatebufferwith10mMETDAadded(0.372g/100mlofbuffer)for10minutesatpH4.2or4.6.
2. Washindistilledwater.
3. ProceedasforthepH9.4method.

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NOTES

OnlythepH4.2sectionsneedcounterstaininginhaematoxylin.SectionsmustbeVERYwellwashedaftersteps3and5toremoveanytracesofprevioussolutions

NOTE:NoresponsibilityisassumedbyTheUniversityofNottinghamortheQueensMedicalCentreNHSTrustforanyinjuryand/ordamagetopersonsorpropertyasamatterofproductsliABIlity,negligenceorotherwise,orfromanyuseoroperationofanymethods,products,instructionsorideascontainedinthematerialherein.ItistheusersresponsibiltytoensurethatallproceduresarecarriedoutaccordingtoappropriateHealthandSafetyrequirements.

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