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).
Fluorochrome公司专业生产荧光金及荧光金抗体。公司拳头产品荧光金于1985年推向市场,广泛用于神经生物学研究。荧光金在紫外线激发下发金黄色光,特点是非常灵敏,不仅能标记胞浆,而且能很好地显示树突分枝,但核和核仁不染色;在胞体内分解慢,甚至在注射后存活2月标记强度仍无明显变化;比较耐紫外线的照射,褪色比较慢;可以经受许多组织学染色处理,因而可以和HRP、免疫组织化学等方法结合。Fluorochrome公司还提供荧光金抗体及红色荧光金,扩大了其应用范围。