Optimized protocol for retinal wholemount preparation for imaging and immunohistochemistry.
Optimized protocol for retinal wholemount preparation for imaging and immunohistochemistry.
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DOI：
10.3791/51018
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发表时间：
2013-12-13
期刊：
影响因子：
--
通讯作者：
Sagdullaev BT
中科院分区：
文献类型：
--
作者：
Ivanova E;Toychiev AH;Yee CW;Sagdullaev BT
在进行免疫组织化学评估时，与脆弱组织一起工作可能是一个复杂的因素。在这里，我们提出了一种方法，利用环支持的亲水性聚四氟乙烯膜在免疫组织化学处理期间为活组织和固定组织提供结构支持，这允许使用各种方案，否则会对组织造成损害。首先，这是通过将荧光标记物大量加载到活视网膜组织中来证明的。该方法可以快速可视化目标结构，而膜支撑在注射过程中保持组织完整性，并且可以轻松转移制备物以进行进一步成像或处理。其次，描述了在用碳二亚胺固定的组织中进行抗体染色的程序。虽然多聚甲醛固定更为常见，但碳二亚胺固定为突触蛋白的可视化提供了更好的底物。碳二亚胺的一个局限性是所得到的固定组织相对脆弱；然而，这是克服使用支持膜。视网膜组织被用来演示这些技术，但它们也可以应用于任何脆弱的组织。
Working with delicate tissue can be a complicating factor when performing immunohistochemical assessment. Here, we present a method that utilizes a ring-supported hydrophilized PTFE membrane to provide structural support to both living and fixed tissue during immunohistochemical processing, which allows for the use of a variety of protocols that would otherwise cause damage to the tissue. First, this is demonstrated with bolus loading of fluorescent markers into living retinal tissue. This method allows for quick visualization of targeted structures, while the membrane support maintains tissue integrity during the injection and allows for easy transfer of the preparation for further imaging or processing. Second, a procedure for antibody staining in tissue fixed with carbodiimide is described. Though paraformaldehyde fixation is more common, carbodiimide fixation provides a superior substrate for the visualization of synaptic proteins. A limitation of carbodiimide is that the resulting fixed tissue is relatively fragile; however, this is overcome with the use of the supporting membrane. Retinal tissue is used to demonstrate these techniques, but they may be applied to any fragile tissue.
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影响因子：
3
作者：
Yee CW;Toychiev AH;Sagdullaev BT
通讯作者：
Sagdullaev BT
影响因子：
3.2
作者：
Dmitrieva, Nina A.;Strang, Christianne E.;Keyser, Kent T.
通讯作者：
Keyser, Kent T.
影响因子：
3
作者：
Toychiev, Abduqodir H.;Sagdullaev, Bakhodir;Sagdullaev, Botir T.
通讯作者：
Sagdullaev, Botir T.
影响因子：
3.9
作者：
Csaba, G;Kovács, P;Pállinger, É
通讯作者：
Pállinger, É
影响因子：
4
作者：
Tymianski, M;Bernstein, GM;Jones, OT
通讯作者：
Jones, OT