Helliot B, Panis B, Busogoro J P, Sobry S, Poumay Y, Raes M, Swennen R, Lepoivre P
Plant Pathology Unit, Gembloux Agricultural University, Passage des Déportés, Gembloux, Belgium
Eur J Histochem. 2007 Apr-Jun;51(2):153-8.
The immunogold-silver staining (IGSS) technique in combination with epi-fluorescence detection was used to localise cucumber mosaic virus (CMV) particles within banana infected tissues. For this purpose, tissue samples (2 mm3) were excised from CMV-infected and highly proliferating meristem cultures of Williams BSJ banana (ITC. 0570, AAA, Cavendish subgroup). These samples were immediately fixed in a 2% paraformaldehyde/0.25% glutaraldehyde mixture, dehydrated in ethanol, and finally embedded in L.R. White resin. Semi-thin sections were cut, mounted on clean treated glass slides and immunostained for CMV particles using gold-labelled secondary antibodies and silver enhancement. Sections were counterstained with basic fuchsin and examined using laser scanning confocal microscopy. Negative controls included immuno-stained samples excised from non-virus infected material as well as infected material on which primary or secondary antibodies were not applied. Images of autofluorescence (in red) and of epi-reflectance of silver-enhanced immunogold particles (in green) were recorded separately and merged, allowing the specific localisation of CMV particles at the cellular level on semi-thin sections of aldehyde-fixed banana tissues. The main advantage of this analytical approach compared to previously published protocols is that it combines a fast staining procedure, stable preparation, a high resolution, and a narrow plane of focus with the flexibility in generation, processing and analysis of images offered by laser scanning confocal microscopy. Finally, the presence of numerous CMV particles within banana meristems constitutes a clear explanation of the very low CMV elimination efficiency when using meristem-tip culture alone.
免疫金银染色（IGSS）技术结合落射荧光检测用于在感染香蕉的组织中定位黄瓜花叶病毒（CMV）颗粒。为此，从威廉姆斯BSJ香蕉（ITC. 0570，AAA，卡文迪什亚组）感染CMV且高度增殖的分生组织培养物中切取组织样本（2立方毫米）。这些样本立即固定在2%多聚甲醛/0.25%戊二醛混合物中，在乙醇中脱水，最后包埋在L.R. White树脂中。切取半薄切片，安装在经处理的干净载玻片上，使用金标记二抗和银增强剂对CMV颗粒进行免疫染色。切片用碱性品红复染，并使用激光扫描共聚焦显微镜检查。阴性对照包括从未感染病毒的材料切取的免疫染色样本以及未应用一抗或二抗的感染材料。分别记录自发荧光图像（红色）和银增强免疫金颗粒的落射反射图像（绿色）并合并，从而在醛固定香蕉组织的半薄切片上在细胞水平特异性定位CMV颗粒。与先前发表的方案相比，这种分析方法的主要优点在于，它将快速染色程序、稳定制备、高分辨率和窄聚焦平面与激光扫描共聚焦显微镜在图像生成、处理和分析方面的灵活性结合在一起。最后，香蕉分生组织中存在大量CMV颗粒清楚地解释了单独使用茎尖培养时CMV消除效率极低的原因。
