Ultrastructural distribution of myosin heavy chain mRNA in cardiac tissue: a comparison of frozen and LR White embedment.
Ultrastructural distribution of myosin heavy chain mRNA in cardiac tissue: a comparison of frozen and LR White embedment.
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DOI：
10.1177/39.8.1856452
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发表时间：
1991-08
期刊：
影响因子：
--
通讯作者：
M. Wenderoth;B. Eisenberg
中科院分区：
文献类型：
--
作者：
M. Wenderoth;B. Eisenberg
电子显微镜 (EM) 原位杂交提供了确定特定 mRNA 与包含该信息编码的蛋白质的细胞器之间的空间关系所需的更高分辨率。 EM 原位杂交用于确定正常心脏组织中肌原纤维的亚细胞肌球蛋白重链 (MHC) mRNA 分布。比较冷冻或丙烯酸包埋组织切片的超微结构完整性和内源 mRNA 含量。从正常兔子心脏切下的乳头肌进行醛固定，冷冻或包埋在 LR White 中。通过间接免疫细胞化学检测无核糖核酸探针、载体序列、同义和反义生物素化核糖核酸探针的EM原位杂交。使用反义探针的标记密度在肌原纤维间空间最高，平均信号是背景的五倍。非特异性正义探针的背景标记始终较低，但不是随机的，在肌原纤维间隙上也具有最高密度的金簇。超低温切片术产生了更高绝对数量的金簇，但切片破碎并破坏了横纹肌形态。 LR White 包埋保持了超微结构的完整性，但给出了较低的绝对信号。幸运的是，MHC mRNA 信息丰富，可以耐受 LR White 敏感性下降。
Electron microscopy (EM) in situ hybridization provides the higher resolution necessary to determine the spatial relationship between a specific mRNA and the organelle containing the protein encoded by that message. EM in situ hybridization was used to determine the subcellular myosin heavy chain (MHC) mRNA distribution with respect to the myofibril in normal cardiac tissue. Sections of frozen or acrylic-embedded tissue were compared for ultrastructural integrity and content of endogenous mRNA. Papillary muscles dissected from hearts of normal rabbits were aldehyde-fixed and either frozen or embedded in LR White. EM in situ hybridization with no riboprobe, vector sequence, same-sense, and anti-sense biotinylated riboprobes was detected by indirect immunocytochemistry. Labeling density using an antisense probe was highest over the intermyofibrillar space, with an average signal five times that of background. Background labeling by nonspecific sense probe was consistently low but not random, also having the highest density of gold clusters over the intermyofibrillar space. Ultracryomicrotomy yielded a higher absolute number of gold clusters, but sections were fragmented and disrupted striated muscle morphology. LR White embedment maintained ultrastructural integrity but gave a lower absolute signal. Fortunately, MHC mRNA is an abundant message and can tolerate the decreased sensitivity of LR White.