Specimen Preparation

Different techniques require that tissue be fixed and processed in a variety of different ways. This in turn influences which mounting media may be used. Different kinds of mounting media require different optical considerations. In particular, the refractive index of the mountant will affect the spatial resolution that can be achieved for a given objective.

3.1.1. Whole-Mount Specimens

Particular care must be taken in mounting relatively large, intact or partially dissected embryos under a coverslip. Although such material may be photographed through a dissection microscope, mounting a specimen under cover-glass and in a mountant with a high refractive index will give a better spatial resolution. It may be desirable to clean slides and cover-glass prior to use (see Subheading 2.3.) and to ensure that the thickness of cover-glass is exactly 0.17 mm where optimal resolution or transmission of, in particular, fluorescent light is required (see Subheading 3.2.6.). Whole-mount specimens are mounted under raised coverslips, which use small drops of silicon grease at each corner of the coverslip as supports. The coverslip is thus held above the specimen, and its final height can be adjusted by carefully applying downward pressure. Without silicon grease supports, delicate embryonic tissue is easily flattened. Excess mountant can be removed using absorbent Postlip paper (Hollingsworth & Vose Co., Winchombe Glos., UK). If a nonsetting mountant (e.g., glycerol) is being used, then the edges of the coverslip can be sealed by painting a heavy layer of nail varnish around the edges of the glass.

Even with the support of a silicon grease plinth, applying too much downward pressure to the specimen is potentially disastrous. Therefore, cleaning the cover-glass above a whole-mount preparation requires care. Dust and grease can be removed by dragging a piece of lens tissue, which has been moistened with a drop of 70-100% alcohol, across its surface.

3.1.2. Mountants

Choice of mountant is influenced by whether the specimen is dehydrated, and affects not only the permanence of the preparation, but also the clarity of the image. Embryos that are small and virtually transparent can be fixed whole or sectioned and cleared with glycerol/PBS, which itself can be used as a mountant. Glycerol remains liquid and cannot be regarded as a reliable permanent mounting medium. By contrast, mountants that set to form a solid matrix are not only more stable over time, but also allow the use of oil immersion objective lenses which give optimal visual resolution. Oil immersion lenses should not be used on anything other than permanent preparations (see Subheading 3.2.4.).

1. Nonpermanent preparations—a convenient mountant, which also clears delicate whole mounts is a mixture of 90% glycerol/0.02% azide in PBS (see Note 1). For fluorescent specimens 2.5% DABCO (1,4-diazobicyclo-[2.2.2]-octane) can be added to this mixture to prevent fading. Stains that use FITC as a fluorochrome should be mounted in a medium with a pH of at least 8.5. For a more durable preparation, the cover-glass can be sealed with nail varnish.

2. Permanent preparations—water-soluble permanent mountants (i.e., which set) usually suffer from shrinkage, which can ruin a specimen. The more viscous they are, the less of a problem this is likely to be. However, viscosity makes the mountant hard to handle. Probably the best aqueous, setting mountant is Gelvatol (Monsanto)/Mowiol 4-88 (Hoechst), which hardens overnight and which can be stored as frozen aliquots (see Subheading 2.4.)

Some tissues can only be cleared adequately by xylene or its less toxic equivalent, Histoclear (National Diagnostics, Hessle Hull, UK), having first been dehydrated through an alcohol series. Some counterstains, such as cresyl violet (Nissl), require that the tissue is dehydrated to xylene/Histoclear. Tissue which to be cleared in this way can be mounted in DePeX or, if fluorescent, in Fluoromount (Sigma), both of which set solid over time.

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