Ultrastructural non-radioactive in situ hybridization of GH mRNA in rat pituitary gland: pre-embedding vs ultra-thin frozen sections vs post-embedding.
Ultrastructural non-radioactive in situ hybridization of GH mRNA in rat pituitary gland: pre-embedding vs ultra-thin frozen sections vs post-embedding.
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大鼠垂体中 GH mRNA 的超微结构非放射性原位杂交：包埋前、超薄冰冻切片与包埋后。
DOI：
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发表时间：
1992
影响因子：
3.2
通讯作者：
G. Morel
中科院分区：
文献类型：
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作者：
D. Le Guellec;A. Trembleau;C. Péchoux;F. Gossard;G. Morel
可以使用三种不同的方法进行超微结构水平的原位杂交：在环氧树脂包埋前的振动切片机切片上，超薄冷冻切片上，或在亲水性树脂（如Lowicryl）包埋的组织超薄切片上。为了比较这三种方法的灵敏度、分辨率和超微结构的保存，我们用合成的寡核苷酸标记在生物素-21 dUTP的3'端，与Gln 45-Ser 54的mRNA密码子互补，在超微结构水平上检测了垂体前叶细胞生长激素（GH）基因的表达。所有这些方法得到了相似的结果：mRNA定位于生长激素细胞的板层内质网。预包埋方法提供了最好的超微结构保存，与酶检测系统的低分辨率和中间的灵敏度。10 pmol/ml的探针浓度足以获得信号。用这种方法，未经预处理的金颗粒不能使用。冷冻切片法的灵敏度最高（探针浓度为4 pmol/ml时观察到信号），但超微结构保存率最低。在超薄Lowicryl切片上，分辨率与冷冻切片法一样高，超微结构保存中等，灵敏度低。这些结果表明，最后一种方法似乎是一个很好的折衷之间的敏感性和超微结构的保护。
In situ hybridization at the ultrastructural level can be carried out using three different methods: on vibratome sections before embedding in epoxy resin, on ultra-thin frozen sections, or on ultra-thin sections of tissues embedded in hydrophilic resin such as Lowicryl. With the purpose of comparing the sensitivity, resolution, and ultrastructural preservation of these three methods, we examined the expression of the growth hormone (GH) gene in anterior pituitary cells by in situ hybridization at the ultrastructural level, using a synthetic oligonucleotide complementary to the codons of the mRNA from Gln 45 to Ser 54 labeled at the 3' end of biotin-21dUTP. All these methods gave similar results: mRNA was located on the lamellar endoplasmic reticulum of somatotrophs. The pre-embedding method gave the best ultrastructural preservation, with low resolution with the enzymatic detection system and an intermediate sensitivity. A probe concentration of 10 pmol/ml was sufficient to obtain a signal. With this method gold particles could not be used without pre-treatment. The frozen section method gave the best sensitivity (a signal was observed with 4 pmol/ml of probe) but the lowest ultrastructural preservation. On ultra-thin Lowicryl sections, resolution was as high as with the frozen-section method, ultrastructural conservation was intermediate, and sensitivity was low. These results indicate that the last method seems to be a good compromise between sensitivity and ultrastructural preservation.
通过超微结构原位杂交可视化胚胎肌肉组织中肌球蛋白重链 mRNA 的分布。
影响因子：
2.7
作者：
Pomeroy,ME;Lawrence,JB;Singer,RH;Billings-Gagliardi,S
通讯作者：
Billings-Gagliardi,S