嗜酸性粒植物视网膜免疫组织化学、西方分析和 Rna 分离的解剖
Summary
本文介绍了一种解剖嗜酸性粒体木偶视网膜的手术方法, 以及免疫组织化学、西方分析和 rna 提取组织处理的方案。
Abstract
五食子性视网膜为研究发育过程中的形态发生过程提供了一个很好的模型系统。本文提出了一种可靠的方法来解剖娇嫩的酒糟体木偶视网膜。我们的手术方法利用现成的微解剖工具来打开小管, 精确地提取眼脑复合物。这些可以固定, 接受免疫组织化学, 视网膜, 然后安装在显微镜幻灯片和成像, 如果目标是检测细胞或亚细胞结构。或者, 不固定的视网膜可以从脑组织中分离出来, 在适当的缓冲液中裂解, 并用于蛋白质凝胶电泳或 mRNA 提取 (分别用于评估蛋白质或基因表达)。掌握所描述的微解剖协议可能需要大量的实践和耐心, 但一旦掌握了该协议, 就可以相对快速地隔离主要是未损坏的视网膜。
Introduction
嗜德维子由大约750个被细胞包围的孔体组成, 细胞排列在蜂窝晶格1、2、3、4 中.每个瘤包含八个光感受器神经元, 四个透镜分泌锥细胞, 和两个主要色素细胞。周围的每个复合材料都是产生色素的晶格细胞和感觉刚毛群。由于其后有丝分裂的性质和陈规定型六角形排列,五合子木偶视网膜提供了一个很好的模型系统, 研究形态发生过程, 包括细胞粘附5,6, 7,8,9,10和凋亡 11,12,13, 14,15.
几个已发表的协议利用气压从五食小狗 16,17,18提取眼脑复合物。这里描述的协议反而利用微解剖工具, 仔细和精确地分离眼睛-大脑复合物, 目的是获得未受损的视网膜组织。如果视网膜被用于形态、蛋白质或基因表达分析, 这一点至关重要, 因为视网膜的损伤会导致细胞压力或死亡, 从而改变细胞表型或基因表达。此外, 经过练习, 6 至10个眼脑复合物可以在10至15分钟内分离, 从而有助于实现在解剖眼组织的年龄和发育阶段最大限度地减少变异性的目标。
下面描述的固定、免疫染色和全安装协议适用于荧光显微镜用五全套眼睛的制备。视网膜可以与针对感兴趣的蛋白质的抗体一起孵育。例如, 抗体对粘附结成分可用于可视化细胞的顶部周长, 以便评估包括细胞类型、形状和排列在内的特征19。在固定之前, 眼睛可以从大脑中分离出来, 用于提取用于西方分析的蛋白质, 或用于 qRT-PCR 或 rna 测序的 RNA。
Protocol
1. 组织制备 建立五合子十字架 (如前面所述 20) 或培养特定的嗜酸性粒菌株, 以获得所需的基因型的脓包。为了确保大量的小鱼巧合地出现, 在营养丰富的食品培养基或标准的食品培养基上大量补充酵母糊, 建立这些苍蝇培养物一式两份。 在 25°c 保持果蝇培养。对于使用 UAS-GAL4系统的交叉, gmr-gal4是一个理想的驱动程序, 表达在幼?…
Representative Results
木偶眼是一种易于接触的组织, 可作为一个很好的模型来研究驱动形态发生的发育过程。在这里, 我们解剖视网膜, 并使用免疫荧光检测在细胞凋亡过程中激活的位粘连 (图 3a, c) 或 dcp-1 caspase (图 3A) (图3)25。这些方法使人们能够清楚地观察细胞在关键的形态发生过程中的作用, 包括原代细胞的吸收和形…
Discussion
这里描述的五食母目解剖方法允许在10至15分钟内分离6到10个眼睛-大脑复合物。然而, 为了掌握解剖技术, 提高解剖质量和速度, 耐心和实践是必不可少的。这种短暂的解剖时间确保了每只眼睛大约处于相同的发育阶段, 减少了数据集中视网膜表型或基因表达的变异性。虽然替代协议可能需要较少的实践掌握, 我们的协议旨在有条不紊地分离微妙的视网膜组织, 同时最大限度地减少撕裂或剪切?…
Declarações
The authors have nothing to disclose.
Acknowledgements
我们感谢扎克鼓和我们的审查人员对手稿的有益评论。这项工作得到了 R15GM114729 的支持。
Materials
| Adobe Photoshop | Adobe | Image processing software | |
| Bamboo splints, 6" | Ted Pella Inc | 116 | |
| Beta mercaptoethanol | Sigma-Aldrich | M3148 | |
| Beta-glycerol phosphate | Sigma-Aldrich | 50020 | |
| Black dissecting dish | Glass petri dish filled to rim with SYLG170 or SYLG184 (colored black with finely ground charcoal powder). Leave at room temperature for 24-48 h to polymerize. | ||
| Blade holder | Fine Science Tools | 10053 | |
| Bovine serum albumin | Sigma-Aldrich | A7906 | |
| cOmplete, EDTA-free protease inhibitor cocktail tablets | Roche | 4693132001 | |
| Confocal microscope (Zeiss LSM 501) | Carl Zeiss | or similar microscope | |
| Diethyl Pyrocarbonate (DEPC) | Sigma-Aldrich | 40718 | |
| Double-sided tape | 3M | 665 | |
| Drosophila food media, nutrient-rich | 7.5% sucrose, 15% glucose, 2.5% agar, 20% brewers yeast, 5% peptone, 0.125% MgSO4.7H2O, 0.125% CaCl2.2H20 | ||
| Drosophila food media, standard | Bloomington Drosophila Stock center cornmeal recipe. (https://bdsc.indiana.edu/information/recipes/bloomfood.html) | ||
| Ethylenediaminetetraacetic acid | Sigma-Aldrich | E6758 | |
| Fixative solution | 4% formadehyde in PBS, pH 7.4. | ||
| Fluorescence microscope (TCS SP5 DM microscope) | Leica Microsystems | or similar microscope | |
| Forceps | Fine Science Tools | 91150-20 | Forceps should be sharpened frequently. |
| Formaldehyde | Thermo Scientific | 28908 | |
| Glass 9-well dishes | Corning | 7220-85 | Also known as 9-well dishes |
| Glass coverslips (22 x 22 mm) | Fisher Scientific | 12-542-B | |
| Glass microscope slides (25 x 75 x 1 mm) | Fisher Scientific | 12-550-413 | |
| Glass petri dish | Corning | 3160-100BO | |
| Glycerol | Sigma-Aldrich | G5516 | |
| Image Studio software version 5.2.5 | LI-COR Biosciences | Image processing software for quantitation of Western blots. | |
| Laemmli sample buffer | Bio-Rad | 161-0737 | 2X concentrated protein sample buffer, supplement with beta mercaptoethanol as per manufacturer's instructions. |
| Lane marker reducing sample buffer | ThermoFisher Scientific | 39000 | 5X concentrated protein sample buffer. |
| Microcentrigure tubes | Axygen | MCT-175-C | |
| Microdissection scissors | Fine Science Tools | 15000-03 | |
| Microwell trays (72 x 10 µL wells) | Nunc | 438733 | |
| Mounting media | 0.5% N-propylgallate and 80% glycerol in PBS | ||
| N-propylgallate | Sigma-Aldrich | P3130 | |
| Nuclease-free PBS (PBS in 0.1% DEPC, pH 7.4) | Add appropriate volume of DEPC to PBS, mix well and incubate overnight at room temperature with constant stirring. Autoclave for at least 20 minutes. Store at 4°C | ||
| PBS (phosphate buffered saline pH 7.4) | Sigma-Aldrich | P5368 | Prepare according to manufacturer's instructions |
| PBS+pi (PBS plus protease and phoshatase inhibitors) | 10mM NaF, 1mM beta-glycerol phosphate and 1mM Na3VO4 in PBS, pH 7.4. | ||
| PBT | 0.15% TritonX and 0.5% bovine serum albumin in PBS, pH 7.4 | ||
| Pin holder | Fine Science Tools | 26016-12 | |
| Primary antibody: goat anti-GAPDH | Imgenex | IMG-3073 | For Western blotting. Used at 1:3000 |
| Primary antibody: rabbit anti-cleaved Dcp-1 | Cell signaling | 9578S | For immunofluorescence. Used at 1:100 |
| Primary antibody: rat anti-DEcad | Developmental Studies Hybridoma Bank | DCAD2 | For immunofluorescence. Used at 1:20 |
| Primary antibody: rat anti-DEcad | DOI: 10.1006/dbio.1994.1287 | DCAD1 | Gift from Tadashi Uemura. Used at 1:100. |
| RNA extration kit: Relia Prep RNA tissue Miniprep kit | Promega | Z6110 | |
| Rnase decontamination reagent (RNase Away) | Molecular BioProducts | 7002 | |
| Scalpel blades | Fine Science Tools | 10050 | Break off small piece of scapel blade and secure in blade holder. |
| Secondary antibody: 488-conjugated donkey anti-rat IgG (H+L) | Jackson ImmunoResearch | 712-545-153 | For immunofluorescence. Used at 1:200 |
| Secondary antibody: cy3-conjugated goat anti-rabbit IgG (H+L) | Jackson ImmunoResearch | 111-165-144 | For immunofluorescence. Used at 1:100 |
| Secondary antibody: HRP-conjugated goat anti-rat IgG (H+L) | Cell Signaling Technology | 7077 | For Western blotting. Used at 1:3000 |
| Secondary antibody: HRP-conjugated rabbit anti-goat IgG (H+L) | Jackson ImmunoResearch | 305-035-003 | For Western blotting. Used at 1:3000 |
| Sodium Chloride | Sigma-Aldrich | S3014 | |
| Sodium Fluoride | Sigma-Aldrich | 215309 | |
| Sodium vanadate | Sigma-Aldrich | 50860 | |
| Spectrophotometer (NanoDrop) | ThermoFisher Scientific | 2000c | |
| Stereo dissecting microscope (M60 or M80) | Leica Microsystems | or similar microscope | |
| Sylgard (black) | Dow Corning | SYLG170 | |
| Sylgard (transparent) | Dow Corning | SYLG184 | Color black with finely ground charcol powder |
| Tissue: Kimwipes | KIMTECH | 34120 | |
| TritonX | Sigma-Aldrich | T8787 | |
| Trizma hydrochloride pH7.5 | Sigma-Aldrich | T5941 | |
| Tungsten needle, fine | Fine Science Tools | 10130-10 | Insert into pin holder |
| Tungsten needle, sturdy | Fine Science Tools | 10130-20 | Insert into pin holder |
| WTLB (western tissue lysis buffer) | 150mM NaCl, 1.5% Triton X-100, 1mM EDTA, 20% glycerol, 10mM NaF, 1mM beta-glycerol phosphate and 1mM Na3VO4 in 50mM Tris-HCl (pH 7.5). Supplement with one cOmplete protease cocktail table per 10 mL solution. | ||
| Yeast paste | (local supermarket) | Approximately 2 tablespoons Fleischmann's ActiveDry Yeast (or similar) dissolved in ~20 mL distilled H2O |
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Citar este artigo
DeAngelis, M. W., Johnson, R. I. Dissection of the Drosophila Pupal Retina for Immunohistochemistry, Western Analysis, and RNA Isolation. J. Vis. Exp. (145), e59299, doi:10.3791/59299 (2019).