Gillot I, Ciapa B, Payan P, De Renzis G, Nicaise G, Sardet C
Unité de Biologie Cellulaire Marine URA 671 CNRS/Paris VI, Villefranche-sur-mer, France.
Histochemistry. 1989;92(6):523-9. doi: 10.1007/BF00524764.
Freeze-substitution was used to study the distribution of calcium in sea urchin eggs, and the validity of the technique was assessed. We followed the fate of both total and exchangeable calcium of sea urchin eggs in two species (Paracentrotus lividus and Arbacia lixula) after the various treatments needed for freeze-substitution and embedding. We compared the calcium content either by X-ray microanalysis of Epon-embedded sections of freeze-substituted eggs (6.2 +/- 0.71 mmoles/kg of Epon-embedded tissue) or by flame spectrometry analysis of living eggs (32.3 +/- 1.30 nmoles/mg protein). After standardization of units, both values lead to similar total calcium content. We also measured the movements of 45Ca from prelabelled eggs. Exchangeable 45Ca as well as total calcium appeared unaffected by the preparative treatment for X-ray microanalysis. In conclusion, our preparative technique for X-ray microanalysis can be considered appropriate for our material and allows us to undertake a subcellular quantification of calcium in various organelles.
采用冷冻置换法研究海胆卵中钙的分布，并评估该技术的有效性。在进行冷冻置换和包埋所需的各种处理后，我们追踪了两种海胆（紫球海胆和刺冠海胆）卵中总钙和可交换钙的去向。我们通过对冷冻置换卵的环氧树脂包埋切片进行X射线微分析（6.2±0.71毫摩尔/千克环氧树脂包埋组织）或对活卵进行火焰光谱分析（32.3±1.30纳摩尔/毫克蛋白质）来比较钙含量。在单位标准化后，两个值得出的总钙含量相似。我们还测量了预标记卵中45Ca的移动情况。可交换45Ca以及总钙似乎不受X射线微分析制备处理的影响。总之，我们用于X射线微分析的制备技术可被认为适用于我们的材料，并使我们能够对各种细胞器中的钙进行亚细胞定量分析。
