ABSTRACT
We describe here a protocol of manual stretching (Fig. 1), modifi ed from Jackson (1998) and from Michalet et al. (1977), which uses fi sh erythrocyte nuclei, a technology which had not been reported before. We have tested it on several species of Antarctic teleosts during several campaigns in the Southern Ocean (ICEFISH, ICOTA-IPEV, REVOLTA-IPEV) and on some eel (Anguilla anguilla) specimens, provided by the BOREA laboratory at MNHN. Since the fi rst step requires a cell suspension, corresponding to a precise nuclear DNA concentration, we used erythrocytes, which are nucleated in teleosts and chondrichthyans, and easy to count with a hemocytometer. Briefl y, freshly sampled erythrocytes are diluted in PBS and mixed in a buffer containing SDS, to denature proteins, dropped onto a Poly-L-Lysine coated slide and dragged
with a large coverslip. Alternatively, the erythrocytes can be embedded in low melting agarose, in order to prevent the DNA fi bers from breaking upon subsequent steps. Plugs are treated with proteinase K to inactivate nucleases and release DNA fi bers and then can be kept for several months in EDTA at +4°C (Michalet et al. 1997), before being melted and spread onto poly-Llysine slides for further experiments. When DNA fi bers are homogeneously stretched on a solid substrate, DNA is readily accessible to probes and detection reagents. FISH (fl uorescence in situ hybridization) using various kinds of probes (repetitive cloned probes, single copy gene probes, BAC clones, etc.) is then possible using very similar protocols to those used for chromosome preparations, with slight modifications. However, detection requires a fl uorescence microscope equipped with a high sensitivity CCD camera.
