FIGURE 1

Fluorescence micrographs of frozen sections of explants of optic nerve and retina (a) or sciatic nerve (b) after 2 d in culture. At least three explants from different mice were examined in each case with similar results. The Bax ^-/- Bak ^-/- explants were compared with wild-type explants from the same litter or from mice of the same age or with Wld ^s explants from mice of the same age. The explant culture method has been described in detail previously.² Briefly, optic nerves, sciatic nerves, and retinas were dissected and either fixed immediately in 2% paraformaldehyde in PBS for 4 h at 4°C or placed in sterile Millipore culture plate inserts (Millicell-CM, 0.4 m 12 mm diameter, Millipore Corporation, Bedford, MA 01730, USA) and cultured in six-well culture plates at 37°C. Culture medium consisted of Neurobasal A, supplemented with B27 (GIBCO BRL, Life Technologies Ltd, Paisley, UK) and penicillin/streptomycin/amphotericin B (Sigma, UK). Explanted tissue was cultured for up to 48 h before being fixed as above. Explants were then cryoprotected in 30% sucrose/PBS before freezing in a 1 : 1 mixture of 30% sucrose/PBS : OCT (TissueTek, Sakura, Netherlands), sectioning at 15 m, immunolabeling, and imaging in a Biorad Radiance scanning confocal microscope (Biorad, Hercules, CA, USA). In (a), mice were 14–21 days old (although similar results were obtained from a Bax ^-/- Bak ^-/- animal examined on the day of birth). A longitudinal section of optic nerve is shown at the top of each panel, and a cross section of the RGC layer of the corresponding retina is shown at the bottom. Retinal cell nuclei were labeled with propidium iodide (red) and apoptotic nuclei (green) were labeled by the TUNEL technique⁸ (Apoptag, Intergen, Oxford, UK). Neurofilaments in the axons of the optic nerves were labeled with a mixture of rabbit antineurofilament antibodies (NA1297, Affiniti Research Ltd, Exeter, UK), followed by fluorescein-conjugated goat anti-rabbit immunoglobulin antibodies (Jackson ImmunoResearch, Pennsylvania, USA), as previously described.² Note that apoptotic RGCs (arrows) are present in both the wild-type and Wld ^s retinal explants but not in the Bax ^-/- Bak ^-/- retinal explants, while fragmented neurofilaments are present in the wild-type and Bax ^-/- Bak ^-/- optic nerve explants but not in Wld ^s optic nerve explants. In (b), mice were 1–2 months old, and the neurofilaments in longitudinal sections of sciatic nerve explants were labeled as in (a). The results are similar to those in the optic nerve explants in (a). Scale bars =10 m