Histological analysis of GFP expression in murine bone
Histological analysis of GFP expression in murine bone
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DOI：
10.1369/jhc.4a6401.2005
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发表时间：
2005-05-01
影响因子：
3.2
通讯作者：
Rowe, DW
中科院分区：
文献类型：
--
作者：
Jiang, X;Kalajzic, Z;Rowe, DW
利用绿色荧光蛋白（GFP）作为视觉组织学标记来识别胚胎发育过程中复杂的细胞相互作用的能力尚未应用于成人组织，因为在石蜡包埋过程中GFP信号的丢失和高自荧光背景，特别是在骨和骨髓切片中。在这里，我们证明了GFP信号在成人脱钙骨的冷冻切片中保存得很好。使用保留组织学关系的磁带转移系统，GFP表达可以与骨生物学研究中使用的标准组织学染色相关。选择双滤光片和强GFP信号，可以很容易地区分至少四种不同的GFP颜色，这些颜色与自动荧光背景明显不同。冷冻切片的另一个优点是更好地保存免疫表位，这使得免疫染色切片具有内源性GFP和来自P-gal标记基因的强lacZ信号的共定位。我们提出了一种记录来自同一组织学切片的多幅图像的方法，该方法允许GFP信号与随后的染色和破坏GFP的程序共定位。举例说明灵活性的双重成像的各种荧光信号在本研究中描述。同样的成像方法可以作为存档、检索和在研究小组之间共享组织学图像的载体。
The power for appreciating complex cellular interactions during embryonic development using green fluorescent protein (GFP) as a visual histological marker has not been applied to adult tissues due to loss of GFP signal during paraffin embedding and a high autofluorescent background, particularly in section of bone and bone marrow. Here we demonstrate that the GFP signal is well preserved in frozen sections of adult decalcified bone. Using a tape-transfer system that preserves histological relationships, GFP expression can be related to standard histological stains used in bone biology research. The choice of a dual-filter cube and a strong GFP signal makes it possible to readily distinguish at least four different GFP colors that are distinctly different from the autofluorescent background. An additional advantage of the frozen sections is better preservation of immunological epitopes that allow colocalization of an immunostained section with an endogenous GFP and a strong lacZ signal emanating from a P-gal marker gene. We present an approach for recording multiple images from the same histological section that allows colocalization of a GFP signal with subsequent stains and procedures that destroy GFP. Examples that illustrate the flexibility for dual imaging of various fluorescent signals are described in this study. The same imaging approach can serve as a vehicle for archiving, retrieving, and sharing histological images among research groups.