本文關(guān)鍵詞： 皮膚 二甲基亞砜 厚組織切片 組織光學(xué)透明 寬場熒光顯微術(shù) 免疫熒光 大鼠　出處：《解剖學(xué)報》2016年05期 　論文類型：期刊論文

【摘要】：目的通過改良免疫熒光染色方案,使皮膚厚切片能在寬場熒光顯微鏡下熒光成像。方法選擇成年雄性SD大鼠5只,經(jīng)Zamboni固定液灌注固定后取后肢足底皮膚,冷凍切片,片厚40μm,行神經(jīng)纖維標(biāo)志物PGP9.5免疫熒光染色。首先比較染色后用梯度甘油溶液透明與不經(jīng)甘油透明對皮膚厚片成像深度的影響;繼之在染色后甘油透明的基礎(chǔ)上,檢測染色前二甲基亞砜(DMSO)處理不同時間(10min、15min和30min)對皮膚背景信號的影響。此外,采用體視顯微鏡檢測梯度甘油透明對切片形態(tài)的影響。最后,用上述DMSO-甘油改良染色法進(jìn)一步對神經(jīng)纖維其他標(biāo)志物NF 200和外周蛋白分別進(jìn)行染色。結(jié)果經(jīng)染色后甘油透明的切片其成像深度顯著大于常規(guī)染色的切片(P0.01);在甘油透明的基礎(chǔ)上,先用DMSO處理能有效降低皮膚背景著色,時間以15min為佳;梯度甘油處理并不會造成皮膚厚片的形態(tài)改變;DMSO-甘油改良染色法也能有效顯示皮膚NF200和外周蛋白免疫反應(yīng)陽性的神經(jīng)纖維。結(jié)論經(jīng)本改良方法染色的皮膚厚片能在寬場熒光顯微鏡下良好成像。
[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.
【作者單位】： 四川大學(xué)華西基礎(chǔ)醫(yī)學(xué)與法醫(yī)學(xué)院組織學(xué)胚胎學(xué)與神經(jīng)生物學(xué)教研室;
【基金】：四川省科技廳科技支撐計劃項目(2013SZ0069)
【分類號】：R361.2
【正文快照】： 觀察皮膚內(nèi)的神經(jīng)纖維,目前常用針對其標(biāo)志蛋白的免疫熒光染色法,以顯示其分布、走行、形態(tài)等。皮膚內(nèi)的神經(jīng)纖維散在分布且多彎曲,因此在較薄的皮膚切片(20μm)中難以觀察到完整、連續(xù)的神經(jīng)纖維,須制備40~60μm的皮膚厚片。普通寬場熒光顯微鏡是許多實驗室的常規(guī)設(shè)備,但對

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