Lizards received intraperitoneal injections of [3H]-thymidine (Amersham; specific activity 5 Ci/mmol). Depending on their survival time, the final dose was different. For survival times of 1.5 h to 3 days, the animals received a single injection with a dose of 5 μCi/g body weight (b. wt.), while for longer survival times (1–12 months) animals received daily injections (5 μCi/g b. wt.) during three consecutive days, up to a total dose of 15 μCi/g b. wt.
For short survival times, we injected four lizards (n = 4) for each survival time (6, 12, 24, and 72 h), except for the 1.5 h survival time, for which we injected five (n = 5). For long survival times (1, 3, 6, and 12 months) we injected 3 animals for each time (n = 3).
Following their corresponding survival time, the animals were deeply anesthetized with Ketolar (ketamine hydrochloride, 0.6 mg/g b. wt.) and perfused with saline (0.9% NaCl), followed by a fixative consisting of 4% PFA and 2% GA. The complete bodies of the lizards were postfixed in the same fixative during 24 h. The brains were removed from the skull, sectioned frontally or longitudinally on a vibratome at 200 μm, postfixed in 2% osmium tetroxide for 2 h, rinsed, dehydrated, and embedded in epoxy resin (Durcupan, Sigma, San Luis, MO, USA). Semithin sections were cut at 1.5 μm with an ultramicrotome (UC6 Ultracut, Leica, Wetzlar, Germany) and mounted on gelatin-coated glass-slides, which were dipped in LM-1 hypercoat emulsion (Amersham), dried in the dark, and stored at 4°C for 30 days. The autoradiographs were developed using standard methods and counterstained with 1% toluidine blue.
For short survival times, we injected four lizards (n = 4) for each survival time (6, 12, 24, and 72 h), except for the 1.5 h survival time, for which we injected five (n = 5). For long survival times (1, 3, 6, and 12 months) we injected 3 animals for each time (n = 3).
Following their corresponding survival time, the animals were deeply anesthetized with Ketolar (ketamine hydrochloride, 0.6 mg/g b. wt.) and perfused with saline (0.9% NaCl), followed by a fixative consisting of 4% PFA and 2% GA. The complete bodies of the lizards were postfixed in the same fixative during 24 h. The brains were removed from the skull, sectioned frontally or longitudinally on a vibratome at 200 μm, postfixed in 2% osmium tetroxide for 2 h, rinsed, dehydrated, and embedded in epoxy resin (Durcupan, Sigma, San Luis, MO, USA). Semithin sections were cut at 1.5 μm with an ultramicrotome (UC6 Ultracut, Leica, Wetzlar, Germany) and mounted on gelatin-coated glass-slides, which were dipped in LM-1 hypercoat emulsion (Amersham), dried in the dark, and stored at 4°C for 30 days. The autoradiographs were developed using standard methods and counterstained with 1% toluidine blue.
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