University of Texas
A technique for spreading maize microsporocyte pachytene chromosomes for phosphotungstic acid staining to allow simultaneous EM visualization of synaptonemal complex lateral and central elements, recombination nodules, and centromeres
--M. P. Maguire
EM viewing of silver-stained spread preparations allows very clear visualization of synaptonemal complex (SC) lateral elements, either as cores in advance of synapsis or in fully synapsed configurations. However, only the lateral elements of the SC stain appropriately with silver, and central elements, recombination nodules, and centromeres remain invisible. Other stains such as phosphotungstic acid (PTA) or uranyl acetate with lead citrate must be used to stain the additional features. But these other stains also stain chromatin and other nuclear components as well, so as to obscure viewing of the SC structures and recombination nodules (RNs). Procedures must be adapted for making spread preparations which effectively remove the undesired components while keeping the SCs and RNs. Since organisms differ somewhat, differing procedures must be adapted for different organisms, notably maize, where meiotic mutants and altered chromosome complements are available for study. These studies are of special interest if late RNs, for example, can be dependably visualized as indicators of crossover positions.
The following is a procedure which allows visualization of maize SC components and RNs and also displays centromeres as rather large spheres. It represents a modification of a procedure for silver staining which was reported in MNL 63:26-27, 1989.
Fresh anthers at pachytene stage are macerated in a deep depression slide in 5µl of a freshly prepared ice cold medium: 1.5% sucrose, 1% polyvinylpyrrolidone and 2.5mM acid EDTA, adjusted to pH 4.7-4.8 with KOH. The suspension is then transferred by pipette to the surface of a 5µl drop of 0.5% Nonidet P40 in another deep depression slide, where it is left for 2 minutes. Then 60µl of an ice cold fix-detergent mixture is added. The fix-detergent mixture consists of 6% paraformaldehyde solution adjusted to pH 8.6 (as described earlier) to which Lipsol detergent has been added to a final concentration of 1.5%. The depression slide is then covered and placed over an ice bath for at least 30 minutes before the suspension is micropipetted to plastic coated microscope slides. Slides are then thoroughly dried, and fixed as described earlier, and stained with PTA at concentrations from 0.1% to 1.0%.
Future adjustments of the procedure are expected to give even clearer
preparations, especially with cautious increases in detergent concentration.
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