For three-dimensional viewing of chromosome positions with respect to each other, squash procedures are unsuitable. The following procedure, which has been developed for paraffin sections, provides consistent staining with differentiation of knob regions for at least premeiotic mitotic chromosomes at prophase (and interphase) stages.

Material is fixed in ethanol-chloroform 3:1 and stored indefinitely in a freezer before use, when it is transferred through three changes of 70% ethanol before paraffin infiltration through a standard graded tertiary butanol series (The laboratory is maintained at 26 C to keep the tertiary butanol from freezing). This is followed by standard paraffin embedding, sectioning and drying of slide-mounted ribbons. Slides are then treated as follows: xylene 3 minutes, a second xylene 2 minutes, 100% tertiary butanol 2 changes 2 minutes each, 95% ethanol 2 minutes, 70% ethanol 2 minutes, 45% acetic acid 5 minutes, a quick wash in boiled distilled water, saturated barium hydroxide at room temperature 10 minutes (saturated barium hydroxide is prepared by adding boiled distilled water at 85 C to barium hydroxide crystals in an Erlenmeyer flask, shaking, filling to the top, tight stoppering and allowing the crystals to settle out overnight; fresh barium hydroxide solution is prepared each day), five washes in boiled distilled water 2 minutes each, 2X SSC at room temperature for one hour, 3 washes in boiled distilled water 2 minutes each, Giemsa stain solution 25 minutes, two washes in Sorensen's buffer at pH 6.8 2 minutes each, one wash boiled distilled water, graded tertiary butanol series (10%, 20%, 40%, 80%) 2 minutes each, 95% ethanol 2 minutes (for destaining-can be varied for suitable degree of destaining), 100% tertiary butanol 2 changes 2 minutes each, 50% tertiary butanol 50% xylene 2 minutes, xylene several changes for a total of at least an hour, mount in permount. This method purposely avoids all air drying because air drying flattens the cells and nuclei. It may be that more consistent staining results are produced in the absence of air drying, so that this type of procedure might also be useful for initially squashed material. Initial fixation of material in ethanol-acetic 3:1 (instead of ethanol-chloroform 3:1) has so far given only poor results. Gibco Giemsa solution at pH 6.8 for use with Gibco Diagnostics chromosome kit #120-6706 has been used.

Marjorie Maguire
 
 

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