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| Untersuchte Arbeit: Seite: 26, Zeilen: 18-32 |
Quelle: Embark 2004 Seite(n): 48, Zeilen: 1-16 |
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| 3.3.2 Preparation of Xenopus oocytes
An adult female Xenopus laevis frog was submersed in one liter of 3- aminobenzoic acid ethyl ester (0.1%) for about 15-30 min (Figure 6A). After the frog was fully anesthetized it was placed on ice for surgery. A small abdominal incision (1 cm) was carried out and a segment of ovary was removed (Figure 6B,C). Subsequently the wound was closed with a reabsorbable suture (Figure 6D). The frog was then kept wet and warm by placing it in a cavity filled by a small amount of warm water to avoid drowning and hypothermia. The ovarial sacs were manually separated into groups of 10-20 oocytes, put into a 15 ml tube and then enzymatically defolliculated by treatment with an OR- 2 (Oocytes-Ringer) (Table 1) solution containing 1-2 mg/ml collagenase A for 2-2.5 h at room temperature (Figure 6E) with gentle agitation. Defolliculation of the oocytes was stopped by washing several times with ND96 (Table 1).This step also removes all detritus permitting oocyte sorting. Oocytes were then sorted using a self-made apparatus (Figure 6F). Only large oocytes (stage V or VI) were selected and stored overnight in a ND96 storage solution at 16°C. |
2.2.2 Preparation of oocytes
An adult female Xenopus laevis frog was submersed in one liter of 3-aminobenzoic acid ethyl ester (0.1%) for about 15-30 min (Fig. 13A). After the frog was fully anesthetized it was placed on ice for surgery. A small abdominal incision (1 cm) was carried out and a segment of ovary was removed (Fig. 13B, C). Subsequently the wound was closed with a reabsorbable suture (Fig. 13D). The frog was then kept wet and warm by placing it in a cavity filled by a small amount of warm water to avoid drowning and hypothermia. The ovarial sacs were manually separated into groups of 10-20 oocytes, put into a 15 ml tube and then enzymatically defolliculated by treatment with an OR-2 (Oocytes-Ringer) solution containing 1-2 mg/ml collagenase A for 2-2.5 h at room temperature (Fig. 13E) with gentle agitation. Defolliculation of the oocytes was stopped by washing several times with ND96. This step also removes all detritus permitting oocyte sorting. Oocytes were then sorted using a self-made apparatus (Fig. 13F). Only large oocytes (stage V or VI) were selected and stored overnight in a ND96 storage solution at 16°C. |
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