Simultaneous detection of EGFP and cell surface markers by fluorescence microscopy in lymphoid tissues
Simultaneous detection of EGFP and cell surface markers by fluorescence microscopy in lymphoid tissues
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DOI：
10.1177/002215540305100102
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发表时间：
2003-01-01
影响因子：
3.2
通讯作者：
Randall, TD
中科院分区：
文献类型：
--
作者：
Kusser, KL;Randall, TD
增强型 GFP (EGFP) 是一种强大的工具，用于可视化标记蛋白和转染细胞，并且可以通过活细胞中的荧光显微镜或流式细胞术轻松检测到。然而，如果细胞完整性因冷冻、切片或透化而破坏，可溶性 EGFP 分子可能会丢失。此外，EGFP 的荧光取决于其构象。因此，固定 EGFP 的固定方案也可能会破坏其作为荧光报告基因的用途。在这里，我们确定了哪些制备鼠淋巴组织的方法固定可溶性 EGFP 蛋白并保留其荧光，同时保持各种免疫学重要分子的抗原性并最好地保留组织的整体形态。我们发现在冷冻前未固定的脾冷冻切片中无法观察到 EGFP。然而，在各种条件下固定的组织冷冻切片中可以观察到强烈的 EGFP 荧光。固定对于固定 EGFP 而不是维持构象很重要，因为在未固定的冰冻切片中，免疫荧光只能检测到极少量的 EGFP。虽然对EGFP荧光影响不大，但固定过程中加入蔗糖更好地保留了固定组织的形态。这些方法还保留了用于识别淋巴组织细胞类型的多种分子的抗原性。
Enhanced GFP (EGFP) is a powerful tool for the visualization of tagged proteins and transfected cells and is easily detected by fluorescence microscopy or flow cytometry in living cells. However, soluble EGFP molecules can be lost if cell integrity is disrupted by freezing, sectioning, or permeablization. Furthermore, the fluorescence of EGFP is dependent on its conformation. Therefore, fixation protocols that immobilize EGFP may also destroy its usefulness as a fluorescent reporter. Here we determined which methods of preparing murine lymphoid tissues immobilized soluble EGFP protein and retained its fluorescence while simultaneously maintaining the antigenicity of various immunologically important molecules and best preserving the overall morphology of the tissues. We found that EGFP could not be visualized in frozen sections of spleen that had not been fixed before freezing. However, robust EGFP fluorescence could be observed in frozen sections of tissues fixed under various conditions. Fixation was important to immobilize EGFP rather than to maintain conformation, because only minimal EGFP could be detected by immunofluorescence in unfixed frozen sections. Although it had little effect on EGFP fluorescence, the inclusion of sucrose during fixation better preserved the morphology of fixed tissues. These methods also preserved the antigenicity of a wide variety of molecules used to identify cell types in lymphoid tissues.