Fluorescence Mounting Medium (Antifade)
来自 : 蚂蚁淘
MaterialsNeeded
- 20mlglassscintillationvial
- Smallstirbar
- Foil
- Glycerol
- 1XPBS
- Pipets
- *P-phenylenediamine(EMDChemicalsInc.Cat#PX0730)
- Carbonate-BicarbonateBuffer(seebelow)
- Wrapaglassscintillationvialwithfoilanddropinasmallstirbar.(PPDislight-sensitive.)
- Witha10mlpipetadd9mlsofglyceroltothevial.
- Withthe1000µlPipetmanadd1mlof1XPBS.
- Placeonstirrerandbeginmixing.
- Weighout10mgofp-phenylenediamineontheMettlerbalance.PPDistoxic.Wearglovesanddon’tinhaleit.
- AddthePPDtothevialandstiruntillitisallinsolution(1-2hrs.).Themediumshouldappearalmostcolorlesstoaslighttintofyellow.IfitisanintenseyellowororangecolorthePPDismostlikelycontaminatedandwillhavebackgroundstaining.
- pHthemountingmediumtoapHof8.0-9.0usingtheCarbonate-Bicarbonatebuffer.pHpaperofrange6.5-10.0shouldbeusedtocheckthepHofthemediumafteradditionof12dropsoftheCarb-Bicarbbufferandstirring.AdditionaldropsofbufferareaddeduntillthedesiredpHisreached.
- Aliquotthemountingmediumandstoreat-70oC.
*FlakesofPPDarelargeandshouldbecrushedCarbonate-BicarbonateBuffer
- Makeupa0.2Msolutionofanhydroussodiumcarbonate(2.12g/100ml)
- Makeupa0.2Msolutionofsodiumbicarbonate(1.68g/100ml)
Take4mlsofA+46mlsofBandbringupto200mlswithDH2O.ThepHwillbe9.2.*Note:IfthePPDiscontaminatedorgoesbad(turnsabrowncolor)itwillstainDNA,soeachpreparationshouldbetested.Checkbylookingatmitoticcellstobesurethatchromosomesarenotstained.
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