DeFelipe J, Fairén A
Instituto Cajal, Madrid, Spain.
J Histochem Cytochem. 1993 May;41(5):769-72. doi: 10.1177/41.5.8468459.
We describe in detail a simple method for flat-embedding that can be subsequently used in correlative light and electron microscopic studies. The method can be applied to any material suitable for electron microscopy and is especially useful for study of the synaptology and ultrastructural characteristics of immunocytochemically or morphologically identified neurons or their processes. We present here an example to show how accurately one can delineate the fine details of a complex axonal arborization impregnated with the Golgi method in the mouse cerebral cortex. Golgi-impregnated sections to be studied at the electron microscopic level are osmicated, dehydrated, infiltrated with Araldite resin, flat-embedded, and identified cells or processes photographed. Serial semi-thin sections (1-2 microns thick) are then cut with an ultramicrotome, examined with the light microscope, and the elements rephotographed. Selected semi-thin sections are then resectioned on the ultramicrotome at 60-70 nm and examined electron microscopically. This method allows the systematic and accurate localization of stained cells and processes throughout the successive steps of the procedure.
我们详细描述了一种用于平面包埋的简单方法，该方法随后可用于相关光镜和电镜研究。该方法可应用于任何适合电子显微镜检查的材料，尤其适用于研究经免疫细胞化学或形态学鉴定的神经元及其突起的突触学和超微结构特征。我们在此给出一个例子，展示在小鼠大脑皮层中，利用高尔基方法浸渍的复杂轴突分支的精细细节能够被精确描绘到何种程度。要在电子显微镜水平进行研究的高尔基浸渍切片，需进行锇酸处理、脱水、用阿拉迪特树脂浸润、平面包埋，然后对已鉴定的细胞或突起进行拍照。接着用超薄切片机切出连续的半薄切片（1 - 2微米厚），用光学显微镜检查，并对这些元件重新拍照。然后在超薄切片机上把选定的半薄切片切成60 - 70纳米厚，进行电子显微镜检查。该方法能够在整个连续步骤中对染色的细胞和突起进行系统且精确的定位。
