Cryofixing single cells and multicellular specimens enhances structure and immunocytochemistry for light microscopy
Cryofixing single cells and multicellular specimens enhances structure and immunocytochemistry for light microscopy
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DOI：
10.1046/j.1365-2818.1996.135417.x
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发表时间：
1996-05-01
期刊：
影响因子：
--
通讯作者：
Hepler, PK
中科院分区：
文献类型：
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作者：
Baskin, TI;Miller, DD;Hepler, PK
人们普遍认为冷冻固定在保存细胞结构方面优于化学固定；然而，冷冻固定的使用主要限于电子显微镜。为了了解冷冻固定是否会改善通过光学显微镜观察到的样品结构或抗原性，我们通过深度冷冻冷冻固定烟草和长花百合花粉管以及紫露草雄蕊毛。经过冷冻置换和甲基丙烯酸丁酯包埋后，我们通过光学显微镜发现冷冻固定相对于化学固定的优越性是显而易见的，与化学固定不同，冷冻固定不会扭曲细胞形态，并且以与活细胞非常相似的形式保留了微管和肌动蛋白阵列。此外，为了进一步测试冷冻固定在光学显微镜中的实用性，我们研究了细胞的外观和抗原性的保留。冷冻多细胞器官。将拟南芥的根进行化学固定或浸入式冷冻，然后嵌入上面使用的可移除甲基丙烯酸酯树脂中。我们发现浸入式冷冻比化学固定更好地保留了细胞形态，并且同样改善了肌动蛋白和微管阵列的外观。通过甲苯胺蓝染色或高分辨率诺马斯基光学进行评估，与化学固定的根相比，浸入冷冻的根的细胞具有更多的细胞质。冰晶形成造成的损伤无法通过光学显微镜分辨，即使是在距离表面 40-75 μm 的根内部。我们认为，骤冷将增强光学显微镜水平上的许多研究，包括多细胞器官的研究，在多细胞器官中，冰晶造成的损害可能没有化学固定造成的人工制品严重。
Cryofixation is widely held to be superior to chemical fixation for preserving cell structure; however, the use of cryofixation has been limited chiefly to electron microscopy, To see if cryofixation would improve sample structure or antigenicity as observed through the light microscope, we cryofixed Nicotiana alata and Lilium longiflorum pollen tubes and Tradescantia virginina stamen hairs by plunge freezing. After freeze-substitution and embedding in butylmethylmethacrylate, we found using the light microscope that the superiority of cryofixation over chemical fixation was obvious, Cryofixation, unlike chemical fixation, did not distort cell morphology and preserved microtubule and actin arrays in a form closely resembling that of living cells.Additionally, to test further the usefulness of cryofixation for light microscopy, we studied the appearance of cells and the retention of antigenicity in a plunge-frozen multicellular organ. Roots of Arabidopsis thaliana were either chemically fixed or plunge frozen, and then embedded in the removable methacrylate resin used above, We found that plunge freezing preserved cell morphology far better than did chemical fixation, and likewise improved the appearance of both actin and microtubule arrays. Plunge-frozen roots also had cells with more life-like cytoplasm than those of chemically fixed roots, as assessed with toluidine-blue staining or high-resolution Nomarski optics, Damage from ice crystal formation could not be resolved through the light microscope, even in the interior of the root, 40-75 mu m from the surface. We suggest that plunge freezing would enhance many investigations at the light microscope level, including those of multicellular organs, where damage from ice crystals may be less severe than artefacts from chemical fixation.