Chromatin fixation

Figure 12.6 Fixation in formalin for less than 6h produces blurry detail and loss of chromatin patterns. Some nuclei appear faded (arrow). 

We have found that certain commonly used buffers are incompatible with electron microscopy in general (Tris) or microscopy of nuclei in particular (HEPES, phosphate). Triethanolamine (TEA) has been widely used as a buffer for glutaraldehyde fixation of isolated chromatin (e.g., Thoma et ai, 1979), but is unsatisfactory for whole nuclei. Sodium cacodylate and PIPES are, in our experience, consistently reliable buffers. 

Nuclei prepared by these methods vary in susceptibility to nuclease digestion and chromatin release, those exposed to polyamines being the most resistant. In choosing a buffering agent, it should be borne in mind that, as mentioned above, Tris interferes with aldehyde fixation and should be avoided for most applications involving subsequent imaging steps. PIPES has been the most suitable buffer in our hands. 

This is a combination of the above method with a formaldehyde fixation protocol (Pringle et al., 1991). We find it advantageous in combination with certain applications of FISH (e.g., for testing the location of the telomeres at the nuclear periphery) because the nuclear architecture is less damaged than in ordinary spreads. However, for silver staining of SCs and immunostaining of SC proteins, this method is not recommended because the SC is masked by the surrounding chromatin. 

Phase-contrast microscopy provides a fast and reliable way to evaluate the onset of sperm chromatin decondensation and the appearance of nuclei assembled in vitro (Fig. 1). After incubation, a IO-/1I aliquot of the incubation mixture should be taken and immediately fixed with an equal volume of 8% (w/v) paraformaldehyde in 154 mM PIPES-NaOH, pH 7.5, for 5 min on ice. After fixation, samples should be diluted with an equal volume of extraction buffer as specified above. Then, 5-ix aliquots of this mixture may then be mounted for viewing between slide and cover slip. To ensure adequate separation between the slide and cover slip, about 2 /xl of melted bee s wax should be deposited onto each corner of the cover slip before mounting. 

Two major techniques have been used to study the dimension of chromosome fibers by electron microscopy. The first is the surface spreading method, in which the cells are spread on a water-air interphase, and all the intracellular components are dispersed. But the chromatin and the spindle remain close together and can be picked up on a grid, which can then be prepared for electron microscopic examination. The second technique is the thin-sectioning method. After fixation the tissue is embedded in plastic and cut less than 1000 A thick. Refer to specialized texts for descriptions of these techniques [119]. The important finding is that with the first method, the dimension of the fibers diameter is estimated to be 200-300 A, whereas it is only 80-180 A with the second. 

Protocol 5.2 describes a methanol-acetone fixation technique (Pisano et al. 1993 Cenci et al. 1994) which results in very good preservation of cell morphology. Fixed cells viewed by phase-contrast optics exhibit most of the structural details that can be seen in live material. This allows analysis of unstained fixed preparations and selection of the most suitable ones for immunostaining. Remarkably, the Y loops, which are usually faint and labile in living preparations, become clearly apparent after this type of fixation. Moreover, this fixation protocol results in excellent microtubule preservation for immunostaining with antitubulin antibodies. The main disadvantage of this technique is a poor preservation of chromosome structure. In most instances, the chromosomes do not exhibit a distinct morphology and tend to coalesce into one or more masses of chromatin. 

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