Pretreatment methods to improve nerve immunostaining in corneas from long-term fixed embryonic quail eyes.
Pretreatment methods to improve nerve immunostaining in corneas from long-term fixed embryonic quail eyes.
复制标题
改善长期固定胚胎鹌鹑眼角膜神经免疫染色的预处理方法。
DOI：
10.1016/s0165-0270(99)00109-0
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发表时间：
1999
影响因子：
3
通讯作者：
Conrad,GW
中科院分区：
文献类型：
--
作者：
Barrett,JE;Wells,DC;Conrad,GW
用保存在固定液中几个月的日本鹌鹑胚胎第16天的角膜进行预处理，以提高角膜中神经丝的免疫染色。以下三种预处理的顺序组合：在盐水中短暂微波加热，然后在37°C下用十二烷基硫酸钠（SDS）提取，然后在37° C下用透明质酸酶消化，与未经预先预处理或采用单一或两步方案的胚胎角膜相比，角膜神经丝蛋白的抗体染色显着增加。在应用所有三种预处理的顺序后，观察到最深的神经染色和增加的细分支数量，以及较低的背景染色。因此，应用三步预处理序列的结果优于应用其任何组分单一预处理或甚至它们中的任何两种的组合的结果。因此，这些发现表明，这三种预处理中的每一种都会产生独特的效果，有利于神经丝蛋白的免疫染色，并且它们的单独效果是独立的和累加的。除了胚胎角膜，三步程序也可能是有用的免疫染色的神经在其他非常微妙的，高度水合的组织含有丰富的细胞外基质。
Pretreatment methods were used to improve neurofilament immunostaining in corneas from embryonic day 16 Japanese quail corneas that had been stored in fixative solution for several months. A sequential combination of the following three pretreatments: brief microwave heating in saline, followed by extraction with sodium dodecyl sulfate (SDS) at 37°C, followed by digestion with hyaluronidase at 37°C, produced significantly increased antibody staining of corneal neurofilament proteins, compared with embryonic corneas subjected to no prior pretreatments or to single or two-step protocols. After applying the sequence of all three pretreatments, darkest nerve staining and increased numbers of fine branches were observed, together with lower background staining. Thus, the result of applying the three-step pretreatment sequence is better than that of applying any of its component single pretreatments or even combinations of any two of them. These findings therefore suggest that each of these three pretreatments causes a unique effect, beneficial to immunostaining of neurofilament proteins, and that their individual effects are independent and additive. In addition to embryonic corneas, the three-step procedure also may be useful for immunostaining of nerves in other very delicate, highly-hydrated tissues containing an abundance of extracellular matrix.