一种可用于500μm脑片的高分辨率细胞内标记技术  

A MODIFED INTRACELLULAR LABELLING TECHNIQUE FOR HIGH-RESOLUTION STAINING OF NEURON IN 500 μm-THICKENESS BRAIN SLICE

作　　者：赵明亮[1,2] 刘国龙[1] 隋建峰[1] 阮怀珍[3] 熊鹰[1] 

机构地区：[1]第三军医大学生理学教研室 [2]武警医学院附属医院脑系科中心,天津300162 [3]第三军医大学神经生物学教研室

出　　处：《中国应用生理学杂志》2007年第2期254-255,256,I0003,共4页Chinese Journal of Applied Physiology

基　　金：国家自然科学基金资助项目(30371532);武警医学院博士启动基金(WPS-2007-10)

摘　　要：目的建立简便高效的、与脑片盲法膜片钳记录相结合的生物胞素细胞内标记染色方法。方法制备大鼠听皮层脑片(500μm),采用盲法脑片膜片钳全细胞记录,泳入生物胞素(0.2%)对记录细胞进行标记,经组织化学显色和甘油封片后,沿显微镜Z轴,每隔30μm拍摄一帧显微数码图像,利用Adobe Photoshop软件对神经元进行三维重建。结果标记的神经元层次清楚,可在光镜下分辨出胞体、轴树突分支、棘突等细微结构,而且非特异性背景染色浅;不需要进行厚脑片的二次切片即可对神经元进行三维重建。结论本方法简便易行,结果可靠,分辨率高,而且对设备要求不高。To develop simple but reliable intracellular labelling method for high-resolution visualization of the fine structure of single neurons in brain slice with thickness of 500 μm. Methods： Biocytin was introduced into neurons in 500 μm-thickness brain slices while blind whole cell recording. Following processed for histochemistry using the avidin-biotin-complex method, stained slices were mounted in glycerol on special glass slides. Labelled cells were digital photomicrographed every 30 μm and reconstructed with Adobe Photoshop software. Results： After histochemistry, limited background staining was produced. The resolution was so high that fine structure, including branching, termination of individual axons and even spines of neurons could be identified in exquisite detail with optic microscope. With the help of software, the neurons of interest could be reconstructed from a stack of photomicrographs. Con-clusion： The modified method provides an easy and reliable approach to revealing the detailed morphological properties of single neu-rons in 500 μm-thickness brain slice. Without requisition of special equipment, it is suited to be broadly applied.

关 键 词：细胞内标记 生物胞素 脑片 膜片钳术 免疫组织化学 

分 类 号：Q189[生物学—神经生物学] R322.8[生物学—普通生物学]