Fluorescent labeling of specimens offers numerous advantages, not the least of which is the use of multiple labels linked to different fluorochromes on the same preparation. Fluorescent specimens can be viewed using a compound microscope fitted (usually) with an epifluorescent lamp (for epi-illumnation, the light is both projected and detected through the objective lens), filter blocks which contain dichroic beam-splitting mirrors, and objectives designed to transmit fluorescence (see Subheading 3.2.1.). The basic principle is to excite a fluorochrome within a specimen with wavelengths of UV light that are optimized with respect to the absorption spectrum of that particular molecule. The resulting fluorescence emission is then filtered according to the appropriate maxima of the emission spectrum for that fluorochrome. Where two fluoro-chromes are being used, filtering can be optimized to ensure that the emission spectra of each label do not overlap.
In most experimental situations, a choice of fluorochromes is available. Where two fluorochromes are being used in the same specimen, it is important that their fluorescence signals can be clearly distinguished (for example, different populations of dye-labeled axons or different immunolabels). It is important to compare the specifications of the excitation and emission filters with the absorption and emission spectra of the fluorochromes chosen. Other factors that should be taken into account are the brightness and photostability of a particular fluorochrome. Some fluorescent dyes survive fixation better than others. For example, for intracellular dye injection, the highly fluorescent 5,6-carboxyfluorescein cannot be fixed whereas the relatively less bright lucifer yellow can be cross-linked by paraformaldehyde fixation.
Some special conditions should be noted for fluorescent samples. Glutaral-dehyde is auto-fluorescent and so its use should be avoided. If there is no alternative to this fixative, its autofluorescence can be countered by treatment with NaBH4. Dyes may be pH sensitive; for example FITC fluorescence yield is significantly reduced if the pH of the mountant is <8.5-9.0. Some counter-stains are autofluorescent (e.g., Feulgen and Nissl stains). Fluorescent latex beads, which can be used as axonal tracers, or as implants for the slow release of a variety of soluble factors, are dissolved by alcohol and xylene (see Note 8). In general, background fluorescence increases with time stored. Background can be reduced by keeping tissue at 4°C, but it is advisable to view and, in particular, photograph fluorescent material as soon as possible. Permanent, nonaqueous mountants tend to transmit more light than glycerol-based mountants decreasing photographic exposure times and hence the risk of photobleaching.
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