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

An improved immunofluorescent staining method enables thick skin sections to be imaged by a wide-field fluorescence microscope

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(P<0.01);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。

Objective To develop a method for immunofluorescent staining in thick skin sections and to enable the thick skin sections to be imaged by a wide-field fluorescence microscope. Methods Five male adult SD rats were perfused with Zamboni＇s fixative. The plantar area skin of hindpaws was obtained from the fixed rat. Cryotome sections were cut with 40μm in thickness. The sections were randomly chosen for pan axonal biomarker PGP 9. 5 immunostaining. Firstly,effect of optical clearing was evaluated by comparing imaging depths of skin sections with or without gradient glycerol treatment after staining. Secondly,other skin sections were incubated with dimethyl sulphoxide（ DMSO） for 10 min,15min and 30 min respectively before staining,with glycerol treatment after staining,in order to assess if DMSO deceases background staining and determine the optimized time. To investigate whether morphologic changes occur after gradient glycerol clearing,other two groups of skin sections were selected,treated with glycerol or PBST and observed under an operating microscope. Finally,by utilization our modified method,i. e.,a combination of DMSO and gradient glycerol treatment,other skin sections were stained with two nerve fiber biomarker NF 200 and Peripherin respectively. Results Imaging depths of skin sections after gradient glycerol treatment were significantly deeper than those without glycerol clearing（ P〈 0. 01）. Treating skin with DMSO drastically decreased the background staining of skin sections and 15 minutes incubation of DMSO was the most appropriate. There was no obvious morphologic change in skin sections after gradient glycerol clearing. With our modified method,NF 200-IR and peripherin-IR fibers were effectively labeled.Conclusion By using our modified method,fluorescent immunoreactivities in the thick skin sections can be imaged effectively under a wide-field fluorescent microscope.