Ultrastructural examination of mouse kidney glomerular capillary loop by sandwich freezing and freeze-substitution
Ultrastructural examination of mouse kidney glomerular capillary loop by sandwich freezing and freeze-substitution
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DOI：
10.1093/jmicro/dfac031
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发表时间：
2022-07-02
期刊：
影响因子：
1.8
通讯作者：
Chibana，Hiroji
中科院分区：
文献类型：
--
作者：
Yamaguchi，Masashi;Takahashi-Nakaguchi，Azusa;Chibana，Hiroji
三明治冷冻法是一种将样品置于两个金属盘之间快速冷冻的方法，已用于观察活酵母和细菌的精细的接近天然的超微结构。最近，已发现该方法可用于保存戊二醛固定的动物和人体组织的细胞图像。本文应用此方法观察了小鼠肾小球毛细血管袢的精细结构。然后进行形态测定，并将结果与通过体内冷冻技术获得的数据进行比较，该技术可以提供最接近活组织天然状态的超微结构。结果表明，戊二醛固定后夹心冷冻-冷冻-置换法所获得的肾小球毛细血管袢的超微结构，无论是在细胞图像质量上，还是在定量形态计量学上，均与活体冷冻法所获得的肾小球毛细血管袢的超微结构接近。结果表明，戊二醛固定后夹心冷冻-冷冻-置换法所获得的超微结构比常规化学固定和室温脱水法以及常规快速冷冻-冷冻-置换法所获得的超微结构更能反映细胞和组织的生存状态。三明治冷冻-冷冻-置换技术应作为常规观察生物标本接近天然超微结构的标准方法。
Sandwich freezing is a method of rapid freezing by sandwiching specimens between two metal disks and has been used for observing exquisite the close-to-native ultrastructure of living yeast and bacteria. Recently, this method has been found to be useful for preserving cell images of glutaraldehyde-fixed animal and human tissues. In the present study, this method was applied to observe the fine structure of mouse glomerular capillary loops. Morphometry was then performed, and the results were compared with the data obtained by an in vivo cryotechnique, which may provide the closest ultrastructure to the native state of living tissue. The results show that the ultrastructure of glomerular capillary loops obtained by sandwich freezing–freeze-substitution after glutaraldehyde fixation was close to that of the ultrastructure obtained by in vivo cryotechnique not only in the quality of cell image but also in quantitative morphometry. They indicate that the ultrastructure obtained by sandwich freezing–freeze-substitution after glutaraldehyde fixation may more closely reflect the living state of cells and tissues than conventional chemical fixation and dehydration at room temperature and conventional rapid freezing–freeze-substitution of excised tissues without glutaraldehyde fixation. Sandwich freezing–freeze-substitution techniques should be used routinely as a standard method for observing the close-to-native ultrastructure of biological specimens.