Question

医学翻译

Immunohistochemistry and confocal laser scanning microscopy

Frozen tissue cryosections (10 m thick) were placed on gelatin-coated slides and incubated for 15 min. with 100 mmol glycine and 0.1% carboxylated bovine serum albumin (Aurion, Wageningen, The Netherlands) in PBS, pH 7.4. Then the cryosections were incubated overnight with the primary antibody against vimentin (clone V9; Sigma, St Louis, MO, USA) in a moist chamber. After ashing in PBS, the preparations were incubated with biotinylated donkey antimouse IgG followed by streptavidin-Cy-2 (Biotrend,
Cologne, Germany). The nuclei were stained with 0.002% 7-aminoactinomycin D (Molecular Probes, Carlsbad, CA, USA). Omission of the primary antibody served as a negative control.
The immunolabelled sections were examined with a Leica TCN-NT laser microscope, equipped with argon/krypton and helium/neon laser. Series of confocal sections were taken through the depth of the tissue samples at 0.5-m intervals. In order to improve image quality and to obtain a high signal/noise ratio each image from the series was signal averaged. After data acquisition, the images were transferred to a Silicon Graphics Indy or Octane workstations (Silicon Graphics, Sunnyvale, CA, USA) for image restoration and reconstruction using Imaris, the multi-channel image processing software (Bitplane, Zürich, Switzerland). The principles of this method and numerous images btained with this technique have been previously published [46, 47]. In double-labelling experiments, cryosections were incubated overnight with the primary polyclonal antibody against c-kit (Dako, Glostrup, Denmark) followed by donkey anti-rabbit IgG coupled with Cy2 (Dianova, Hamburg, Germany). After repeated washes in PBS, the preparations were incubated with antivimentin antibodies directly coupled with Cy3. Myofibrils were stained for F-actin with Alexa 633-conjugated phalloidin. The nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI) (Molecular Probes). All preparations were mounted in Mowiol (Hoechst, Frankfurt, Germany). Classical immunocytochemistry was performed on fomalin-fixed, paraffin-embedded tissue sections by the avidin-biotin peroxidase complex method, as previously reported [10]. The primary antibodies used were: CD34 – monoclonal, 1: 100, clone QBEND 10 (Biogenex, San Ramon, CA, USA) and vimentin, monoclonal, 1: 100, clone V-9 (Biogenex).

Answer 1

免疫组织化学和激光共聚焦显微镜扫描

冰冻组织cryosections(10米厚)被放置在gelatin-coated幻灯片和孵化15 min.中和与甘氨酸和0.1%的羧基化咪唑镇静剂100牛血清白蛋白(Aurion成功,荷兰)在PBS,pH值7.4。然后cryosections一夜之间被孵化与原抗体攻击vimentin(克隆客V9;西格码、圣路易斯、钼、美国)在一个潮湿的室。在PBS灰后,随即与缺氧antimouse IgG孵化驴,紧随其后的是streptavidin-Cy-2(Biotrend,

科隆,德国)。细胞核沾满0.002% 7-aminoactinomycin D(分子探针,巴斯、钙、美国)。省略的主要抗体阴性对照担任。

immunolabelled检查的部分用莱卡TCN-NT激光显微镜,配备氩、氦氖/ /氪激光。采取了一系列共焦段通过组织样本的深度0.5 -m间隔。为了改善图像质量,以获得一个高信噪比每一图像信号从系列是平均值。在数据采集、图像转移到一个硅图形印或辛烷值工作站(硅、钙、图形,桑尼维尔美国使用的图像恢复与重建Imaris、多渠道的图像处理软件(Bitplane,苏黎世瑞士)。方法的基本原理和无数的图像btained用这种技术已经以前发表的[第四十六条、第四十七条]。在double-labelling实验,cryosections一夜之间被孵化与原多克隆抗体对c-kit(Dako,Glostrup、丹麦),其次为驴anti-rabbit IgG加上Cy2(Dianova、汉堡、德国)。在经过反复冲洗PBS,准备工作…antivimentin抗体直接与Cy3加上。Myofibrils是为微染,633 -共轭phalloidin灼。细胞核沾满4、6-diamidino-2-phenylindole(金)(分子探针)。所有准备被安装在Mowiol(Hoechst、法兰克福、德国)。经典的免疫细胞化学方法对其进行了fomalin-fixed,paraffin-embedded组织部门avidin-biotin过氧化物酶的复合形法,如前所报道[10]。主要的抗体为:CD34 -单克隆,(2):100 -,克隆QBEND 10(Biogenex,圣罗曼、钙、美国)和vimentin,单克隆,(2):100 -,克隆V-9(Biogenex)。

Answer 2

免疫组织化学和激光共聚焦扫描显微镜冰冻组织冰冻切片（10米厚）被放置在明胶涂层幻灯片和培养15分钟。100甘氨酸和0.1%羧基牛血清白蛋白（奥利昂，瓦赫宁根大学，荷兰）型，PH值7.4。然后冰冻切片孵育过夜的主要对波形蛋白抗体（克隆模式；西格玛，圣路易斯，钼，美国）在潮湿室。灰化后在公共电视网，准备培养生物驴抗抗体其次是streptavidin-cy-2（biotrend，科隆，德国）。细胞核染色与0.002%7-aminoactinomycin丁（分子探针，卡尔斯巴德，加利福尼亚州，美国）。遗漏的主要抗体作为阴性对照。该immunolabelled切片检查与tcn-nt徕卡激光显微镜，配备氩氪和氦氖激光。系列共聚焦部分采取了通过深入的组织样本在0.5 -米间隔。为了提高图像质量和获得高信噪比每个图像从一系列信号平均。经过数据采集，图像被转移到一个硅谷图形工作站印或辛烷值（硅谷图形公司，桑尼维尔，加利福尼亚州，美国）的图像恢复和重建使用imaris，多通道图像处理软件（平面，与ü丰富，瑞士）。该方法的原理和许多图像获得这一技术已先前公布的[ 46，47 ]。在标记的实验中，冰冻切片孵育过夜的原发性多克隆抗体（抗c - kit，丹麦，丹麦）驴抗兔抗体之后，加上接触件（dianova，汉堡，德国）。经过反复冲洗，硫化铅，准备孵育antivimentin抗体直接耦合与Cy 3。肌原纤维染色的F -肌动蛋白与灼633-conjugated鬼笔环肽。细胞核染色与4,6-diamidino-2-phenylindole（吲哚）（分子探针）。所有的准备工作都安装在mowiol（赫斯特，法兰克福，德国）。经典的免疫细胞化学进行fomalin-fixed，石蜡切片中的卵白素一生物素过氧化物酶复合物的方法，如以前所报告[ 10]。主要是：CD 34单克隆抗体用于–：100，1，10（biogenex克隆qbend，圣拉蒙，加利福尼亚州，美国）和波形蛋白，单克隆，1 :100，克隆v-9（biogenex）。