一种用于宽场荧光显微镜下成像的皮肤厚片免疫荧光染色方法

发布时间：2018-02-04 23:26

本文关键词： 皮肤二甲基亚砜厚组织切片组织光学透明宽场荧光显微术免疫荧光大鼠　出处：《解剖学报》2016年05期 　论文类型：期刊论文

【摘要】：目的通过改良免疫荧光染色方案,使皮肤厚切片能在宽场荧光显微镜下荧光成像。方法选择成年雄性SD大鼠5只,经Zamboni固定液灌注固定后取后肢足底皮肤,冷冻切片,片厚40μm,行神经纤维标志物PGP9.5免疫荧光染色。首先比较染色后用梯度甘油溶液透明与不经甘油透明对皮肤厚片成像深度的影响;继之在染色后甘油透明的基础上,检测染色前二甲基亚砜(DMSO)处理不同时间(10min、15min和30min)对皮肤背景信号的影响。此外,采用体视显微镜检测梯度甘油透明对切片形态的影响。最后,用上述DMSO-甘油改良染色法进一步对神经纤维其他标志物NF 200和外周蛋白分别进行染色。结果经染色后甘油透明的切片其成像深度显著大于常规染色的切片(P0.01);在甘油透明的基础上,先用DMSO处理能有效降低皮肤背景着色,时间以15min为佳;梯度甘油处理并不会造成皮肤厚片的形态改变;DMSO-甘油改良染色法也能有效显示皮肤NF200和外周蛋白免疫反应阳性的神经纤维。结论经本改良方法染色的皮肤厚片能在宽场荧光显微镜下良好成像。
[Abstract]:Objective to improve the immunofluorescence staining method to make thick sections of skin imaging under a wide field fluorescence microscope. Methods 5 adult male SD rats were selected. The foot skin of the hind limb was taken after the immobilization with Zamboni fixative, and the thickness of the slice was 40 渭 m. The nerve fiber marker PGP9.5 immunofluorescence staining was performed. Firstly, the effects of gradient glycerol solution transparency and non-glycerol transparency on the depth of skin thickness were compared. Then on the basis of glycerol transparency after dyeing, the effect of DMSO-DMSO on the background signal of skin was detected at different time (10 min, 15 min and 30 min). Stereoscopic microscope was used to detect the effect of gradient glycerol transparency on the morphology of sections. Finally. Further application of DMSO-glycerol modified staining method to NF in nerve fibers. Results the imaging depth of transparent sections of glycerol after staining was significantly higher than that of conventional stained sections (P < 0.05). P0.01; On the basis of glycerol transparency, DMSO treatment can effectively reduce the skin background coloring, and the best time is 15 minutes. Gradient glycerol treatment did not result in the morphological changes of the thick skin tablets. DMSO-glycerol modified staining method can also effectively display NF200 and peripheral protein immunoreactive nerve fibers. Like.
【作者单位】：四川大学华西基础医学与法医学院组织学胚胎学与神经生物学教研室;
【基金】：四川省科技厅科技支撑计划项目(2013SZ0069)
【分类号】：R361.2
【正文快照】： 观察皮肤内的神经纤维,目前常用针对其标志蛋白的免疫荧光染色法,以显示其分布、走行、形态等。皮肤内的神经纤维散在分布且多弯曲,因此在较薄的皮肤切片(20μm)中难以观察到完整、连续的神经纤维,须制备40~60μm的皮肤厚片。普通宽场荧光显微镜是许多实验室的常规设备,但对

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