Ginsbach C, Fahimi H D
Histochemistry. 1987;86(3):241-8. doi: 10.1007/BF00490254.
Filipin is used for ultrastructural cytochemical localization of cholesterol in biological membranes. It binds to unesterified 3 beta-hydroxy-sterols forming 25 nm complexes which are readily recognized in freeze-fracture replicas. Since most investigations with filipin have been performed in isolated cells (tissue culture, cell suspensions etc.) we have investigated the conditions for reproducible labeling of cholesterol in membranes of parenchymatous organs. Vibratome sections of rat kidney fixed by glutaraldehyde perfusion were incubated in filipin and freeze-fracture replicas were prepared using standard techniques. The concentration of filipin, the thickness of vibratome sections and the incubation time and temperature were varied over a wide range. Optimal results were obtained with 50 micron thick tissue slices incubated in 400 micrograms/ml of filipin for 46 h at room temperature. Under these conditions lysosomes were consistently labeled while mitochondria and the endoplasmatic reticulum were negative. Peroxisomes showed a little or no labeling at all while the nuclear envelope was heavily labeled in some cells being negative in others. The method described here should be useful in investigation of the role of cholesterol in function of biological membranes in parenchymatous organs and compact tissues.
菲律宾菌素用于生物膜中胆固醇的超微结构细胞化学定位。它与未酯化的3β-羟基甾醇结合，形成25纳米的复合物，在冷冻断裂复制品中很容易识别。由于大多数关于菲律宾菌素的研究都是在分离的细胞（组织培养、细胞悬液等）中进行的，我们研究了在实质器官膜中可重复标记胆固醇的条件。通过戊二醛灌注固定的大鼠肾脏振动切片在菲律宾菌素中孵育，并用标准技术制备冷冻断裂复制品。菲律宾菌素的浓度、振动切片的厚度、孵育时间和温度在很宽的范围内变化。在室温下，将50微米厚的组织切片在400微克/毫升的菲律宾菌素中孵育46小时可获得最佳结果。在这些条件下，溶酶体始终被标记，而线粒体和内质网呈阴性。过氧化物酶体显示很少或没有标记，而核膜在一些细胞中被大量标记，在另一些细胞中呈阴性。这里描述的方法在研究胆固醇在实质器官和致密组织中生物膜功能中的作用方面应该是有用的。
