A10%solutionofFluoro-Rubyismadebydissolving10mgofFluoro-Rubydrypowderin100µLofdistilledwater.Itisthendeliveredviaintracranialstereotaxicinjectionusinga1uLHamiltonmicrosyringetopressureinjectthetracer.Injectionvolumestypicallyrangefrom0.02-0.1µLandaregraduallyinjectedovera10-15minuteinterval.Theanimalisallowedtorecoverandissacrificedtypically4-14dayslater.
Theanimalisthenperfusedwithneutralbufferedformaldehyde(10%formalinor4%paraformaldehydein0.1Mneutralphosphatebuffer.)Thebrainisremovedandpostfixedforatleastovernightinthesamefixativesolutionplus20%sucroseforcryo-protection.Thebrainisthencutonafreezingslidingmicrotomeorcryostatintosectionsusuallybetween20and40micronsinthickness.
Thetissuesectionsaremountedongelatincoatedslidesandareairdriedonaslidewarmerat50-60degreesCentigradeforatleastonehalfhour.ThedryslidesarethentransferredtoxyleneclearingsolutionforatleastoneminuteandthencoverslippedwithDPXmountingmedia.TheslidescanthenbeexaminedunderanepifluorescentmicroscopeusingafiltersuitableforvisualizingTRITC(greenlightexcitation).
