Name: dissection of the drosophila pupal retina for immunohistochemistry, western analysis, and rna isolation
Uploaded: 2019-03-15T15:00:00
Description: Watch this Scientific Journal Video about Dissection of the Drosophila Pupal Retina for Immunohistochemistry, Western Analysis, and RNA Isolation at JoVE.com

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。

1. 组织制备
<ol><li>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。</li>
	<li>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?...

<ol>
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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?...

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。

<table border="0" cellpadding="0" cellspacing="0" style="width:655px;" width="655">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:145px;">Adobe Photoshop</td>
			<td style="width:131px;">Adobe</td>
			<td style="width:95px;"></td>
			<td style="width:285px;">Image processing software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:145px;">Bamboo splints, 6&#34;&#160;</td>
			<td style="width:131px;">Ted Pella Inc</td>
			<td style="width:95px;">116</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:145px;">Beta mercaptoethanol</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td style="width:95px;">M3148</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:145px;">Beta-glycerol phosphate</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td style="width:95px;">50020</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="90">
			<td height="90" style="height:91px;width:145px;">Black dissecting dish</td>
			<td style="width:131px;"></td>
			<td style="width:95px;"></td>
			<td style="width:285px;">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.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:145px;">Blade holder</td>
			<td style="width:131px;">Fine Science Tools</td>
			<td style="width:95px;">10053</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:145px;">Bovine serum albumin</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td style="width:95px;">A7906</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:85px;width:145px;">cOmplete, EDTA-free protease inhibitor cocktail tablets</td>
			<td style="width:131px;">Roche</td>
			<td style="width:95px;">4693132001</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:145px;">Confocal microscope (Zeiss LSM 501)</td>
			<td style="width:131px;">Carl Zeiss</td>
			<td style="width:95px;"></td>
			<td style="width:285px;">or similar microscope</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:145px;">Diethyl Pyrocarbonate (DEPC)</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td style="width:95px;">40718</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:145px;">Double-sided tape</td>
			<td style="width:131px;">3M</td>
			<td style="width:95px;">665</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="67">
			<td height="67" style="height:67px;width:145px;">Drosophila food media, nutrient-rich&#160;</td>
			<td style="width:131px;"></td>
			<td style="width:95px;"></td>
			<td style="width:285px;">7.5% sucrose, 15% glucose, 2.5% agar, 20% brewers yeast, 5% peptone, 0.125% MgSO4.7H2O, 0.125% CaCl2.2H20</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:85px;width:145px;">Drosophila food media, standard</td>
			<td style="width:131px;"></td>
			<td style="width:95px;"></td>
			<td style="width:285px;">Bloomington Drosophila Stock center cornmeal recipe.&#160; (https://bdsc.indiana.edu/information/recipes/bloomfood.html)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:145px;">Ethylenediaminetetraacetic acid</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td style="width:95px;">E6758</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:145px;">Fixative solution</td>
			<td style="width:131px;"></td>
			<td style="width:95px;"></td>
			<td style="width:285px;">4% formadehyde in PBS, pH 7.4.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:145px;">Fluorescence microscope (TCS SP5 DM microscope)</td>
			<td style="width:131px;">Leica Microsystems</td>
			<td style="width:95px;"></td>
			<td style="width:285px;">or similar microscope</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:145px;">Forceps&#160;</td>
			<td style="width:131px;">Fine Science Tools</td>
			<td style="width:95px;">91150-20</td>
			<td style="width:285px;">Forceps should be sharpened frequently.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:145px;">Formaldehyde</td>
			<td style="width:131px;">Thermo Scientific</td>
			<td style="width:95px;">28908</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:145px;">Glass 9-well dishes&#160;</td>
			<td style="width:131px;">Corning</td>
			<td style="width:95px;">7220-85</td>
			<td style="width:285px;">Also known as 9-well dishes&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:145px;">Glass coverslips (22 x 22 mm)</td>
			<td style="width:131px;">Fisher Scientific</td>
			<td style="width:95px;">12-542-B</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:145px;">Glass microscope slides (25 x 75 x 1 mm)</td>
			<td style="width:131px;">Fisher Scientific</td>
			<td style="width:95px;">12-550-413</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:145px;">Glass petri dish</td>
			<td style="width:131px;">Corning</td>
			<td style="width:95px;">3160-100BO</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:145px;">Glycerol</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td style="width:95px;">G5516</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:145px;">Image Studio software version 5.2.5</td>
			<td style="width:131px;">LI-COR Biosciences</td>
			<td style="width:95px;"></td>
			<td style="width:285px;">Image processing software for quantitation of Western blots.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:145px;">Laemmli sample buffer</td>
			<td style="width:131px;">Bio-Rad</td>
			<td style="width:95px;">161-0737</td>
			<td style="width:285px;">2X concentrated protein sample buffer, supplement with beta mercaptoethanol as per manufacturer&#39;s instructions.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:145px;">Lane marker reducing sample buffer&#160;</td>
			<td style="width:131px;">ThermoFisher Scientific</td>
			<td style="width:95px;">39000</td>
			<td style="width:285px;">5X concentrated protein sample buffer.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:145px;">Microcentrigure tubes&#160;</td>
			<td style="width:131px;">Axygen</td>
			<td style="width:95px;">MCT-175-C</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:145px;">Microdissection scissors&#160;</td>
			<td style="width:131px;">Fine Science Tools</td>
			<td style="width:95px;">15000-03</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:145px;">Microwell trays (72 x 10 &#181;L wells)</td>
			<td style="width:131px;">Nunc</td>
			<td style="width:95px;">438733</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:145px;">Mounting media</td>
			<td style="width:131px;"></td>
			<td style="width:95px;"></td>
			<td style="width:285px;">0.5% N-propylgallate and 80% glycerol in PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:145px;">N-propylgallate</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td style="width:95px;">P3130</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:145px;">Nuclease-free PBS (PBS in 0.1% DEPC, pH 7.4)</td>
			<td style="width:131px;"></td>
			<td style="width:95px;"></td>
			<td style="width:285px;">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&#176;C</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:145px;">PBS (phosphate buffered saline pH 7.4)</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td style="width:95px;">P5368</td>
			<td style="width:285px;">Prepare according to manufacturer&#39;s instructions</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:85px;width:145px;">PBS+pi (PBS plus protease and phoshatase inhibitors)</td>
			<td style="width:131px;"></td>
			<td style="width:95px;"></td>
			<td style="width:285px;">10mM NaF, 1mM beta-glycerol phosphate and 1mM Na3VO4 in PBS, pH 7.4.&#160;&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:145px;">PBT</td>
			<td style="width:131px;"></td>
			<td style="width:95px;"></td>
			<td style="width:285px;">0.15% TritonX and 0.5% bovine serum albumin in PBS, pH 7.4</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:145px;">Pin holder</td>
			<td style="width:131px;">Fine Science Tools</td>
			<td style="width:95px;">26016-12</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:145px;">Primary antibody: goat anti-GAPDH</td>
			<td style="width:131px;">Imgenex</td>
			<td style="width:95px;">IMG-3073</td>
			<td style="width:285px;">For Western blotting. Used at 1:3000</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:145px;">Primary antibody: rabbit anti-cleaved Dcp-1</td>
			<td style="width:131px;">Cell signaling</td>
			<td style="width:95px;">9578S</td>
			<td style="width:285px;">For immunofluorescence. Used at 1:100</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:145px;">Primary antibody: rat anti-DEcad</td>
			<td style="width:131px;">Developmental Studies Hybridoma Bank</td>
			<td style="width:95px;">DCAD2</td>
			<td style="width:285px;">For immunofluorescence. Used at 1:20</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:145px;">Primary antibody: rat anti-DEcad</td>
			<td style="width:131px;">DOI: 10.1006/dbio.1994.1287</td>
			<td style="width:95px;">DCAD1</td>
			<td style="width:285px;">&#160;Gift from Tadashi Uemura. Used at 1:100.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:145px;">RNA extration kit: Relia Prep RNA tissue Miniprep kit&#160;</td>
			<td style="width:131px;">Promega</td>
			<td style="width:95px;">Z6110</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:85px;width:145px;">Rnase decontamination reagent (RNase Away)</td>
			<td style="width:131px;">Molecular BioProducts</td>
			<td style="width:95px;">7002</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:145px;">Scalpel blades</td>
			<td style="width:131px;">Fine Science Tools</td>
			<td style="width:95px;">10050</td>
			<td style="width:285px;">Break off small piece of scapel blade and secure in blade holder.</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:85px;width:145px;">Secondary antibody: 488-conjugated&#160; donkey anti-rat IgG (H+L)</td>
			<td style="width:131px;">Jackson ImmunoResearch</td>
			<td style="width:95px;">712-545-153</td>
			<td style="width:285px;">For immunofluorescence. Used at 1:200</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:145px;">Secondary antibody: cy3-conjugated goat anti-rabbit IgG (H+L)</td>
			<td style="width:131px;">Jackson ImmunoResearch</td>
			<td style="width:95px;">111-165-144</td>
			<td style="width:285px;">For immunofluorescence. Used at 1:100</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:85px;width:145px;">Secondary antibody: HRP-conjugated goat anti-rat IgG (H+L)</td>
			<td style="width:131px;">Cell Signaling Technology</td>
			<td style="width:95px;">7077</td>
			<td style="width:285px;">For Western blotting. Used at 1:3000</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:85px;width:145px;">Secondary antibody: HRP-conjugated rabbit anti-goat IgG (H+L)</td>
			<td style="width:131px;">Jackson ImmunoResearch</td>
			<td style="width:95px;">305-035-003</td>
			<td style="width:285px;">For Western blotting. Used at 1:3000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:145px;">Sodium Chloride</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td style="width:95px;">S3014</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:145px;">Sodium Fluoride</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td style="width:95px;">215309</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:145px;">Sodium vanadate</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td style="width:95px;">50860</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:145px;">Spectrophotometer (NanoDrop)</td>
			<td style="width:131px;">ThermoFisher Scientific</td>
			<td style="width:95px;">2000c&#160;</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:145px;">Stereo dissecting microscope (M60 or M80)</td>
			<td style="width:131px;">Leica Microsystems</td>
			<td style="width:95px;"></td>
			<td style="width:285px;">or similar microscope</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:145px;">Sylgard (black)</td>
			<td style="width:131px;">Dow Corning</td>
			<td style="width:95px;">SYLG170</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:145px;">Sylgard (transparent)</td>
			<td style="width:131px;">Dow Corning</td>
			<td style="width:95px;">SYLG184</td>
			<td style="width:285px;">Color black with finely ground charcol powder</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:145px;">Tissue: Kimwipes</td>
			<td style="width:131px;">KIMTECH</td>
			<td style="width:95px;">34120</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:145px;">TritonX</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td style="width:95px;">T8787</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:145px;">Trizma hydrochloride pH7.5</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td style="width:95px;">T5941</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:145px;">Tungsten needle, fine</td>
			<td style="width:131px;">Fine Science Tools</td>
			<td style="width:95px;">10130-10</td>
			<td style="width:285px;">Insert into pin holder</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:145px;">Tungsten needle, sturdy</td>
			<td style="width:131px;">Fine Science Tools</td>
			<td style="width:95px;">10130-20</td>
			<td style="width:285px;">Insert into pin holder</td>
		</tr>
		<tr height="130">
			<td height="130" style="height:130px;width:145px;">WTLB (western tissue lysis buffer)</td>
			<td style="width:131px;"></td>
			<td style="width:95px;"></td>
			<td style="width:285px;">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.</td>
		</tr>
		<tr height="67">
			<td height="67" style="height:67px;width:145px;">Yeast paste</td>
			<td style="width:131px;">(local supermarket)</td>
			<td style="width:95px;"></td>
			<td style="width:285px;">Approximately 2 tablespoons Fleischmann&#39;s ActiveDry Yeast (or similar) dissolved in ~20 mL distilled H2O</td>
		</tr>
	</tbody>
</table>

dissection of the drosophila pupal retina for immunohistochemistry, western analysis, and rna isolation

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。

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...

Watch this Scientific Journal Video about Dissection of the Drosophila Pupal Retina for Immunohistochemistry, Western Analysis, and RNA Isolation at JoVE.com

Dissection of the Drosophila Pupal Retina for Immunohistochemistry, Western Analysis, and RNA Isolation

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。

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

The Drosophila pupal retina provides an excellent model system for the study of morphogenetic processes during development. In this paper, we present a ...

该协议非常重要，因为这是一种有效的方法来分离多下游应用的果蝇幼眼组织。这项技术的主要优点是，它允许快速解剖大量的幼眼，而不会损害组织。果蝇幼崽视网膜的解剖对新手来说将是具有挑战性的。实践技术是提高解剖速度和结果质量的最佳方法。将果蝇放在黑色解剖盘上后，将一块新鲜的双面胶带放在解剖盘上，远离幼崽，并使用一对钳子小心地将幼崽侧放在胶带上。将盘子放在立体显微镜下，用钳子去除每个幼崽的蛋白石。使用微切除剪刀将每个幼崽皮盒切成片，使其被剥开以露出头部、胸部和前腹部部分，并固定到双面胶带的幼崽箱的边缘。用锋利的钳子刺穿每个幼崽的腹部，然后从幼崽的皮箱中取出幼崽。将幼崽放在解剖盘上，远离胶带，用 400 微升的冰冷 PBS 盖住幼崽。用钳子抓住腹部的每一个，并使用微切除剪刀，使一个干净的横截面切口通过胸部，将幼崽切割为两半。使用两对细钳，抓住每个胸皮的切边，并逐渐撕开胸部和头部胶囊，暴露眼脑复杂和周围的脂肪组织。然后，在不抓住组织的情况下，用钳子引导每个半透明、白色、哑铃形的眼脑复合物，远离头胶囊的残余物。解剖6至10个pae后，用干净的剃须刀刀片将微管尖端切成直径约1毫米，然后用PBS和脂肪的混合物润滑尖端。使用润滑的移液器尖端将眼脑复合物转移到冰上含有 400 微升 PBS 的九井玻璃盘中的一个井中，然后将 20 微升 PBS 中的组织转移到至少 250 微升的固定剂中，在冰上孵育 35 分钟。在固定结束时，使用相同的移液器尖端将组织转移到第三个井中，该井含有 400 微升新鲜 PBS，在冰上进行 5 到 10 分钟。要阻止任何非特异性抗体结合，将眼脑复合物转移到400微升PBS加Triton X中，在冰上孵育10至60分钟。在孵化结束时，将原抗体溶液的10微升等分液放入72孔微井板的必要孔数中，并使用P10空气位移微移子，用PBS加Triton X润滑经过改良的0.5毫米尖端，将不超过5个眼脑复合物和不超过3微升的PBS转移到每口抗体溶液中。在4摄氏度下过夜孵育后，每次洗涤5至10分钟，在400微升PBS加Triton X的一个井中清洗样品，5至10分钟。第三次洗涤后，将组织转移到100微升适当的二次抗体溶液中，在室温下进行两个小时的保护，然后用3个5到10分钟的PBS微升加Triton X和1个PBS洗涤。然后，在200微升安装介质中平衡样品一至两个小时。在孵化结束时，将样品转移到显微镜幻灯片上，并使用坚固的钨针将眼脑复合物固定在解剖显微镜下的幻灯片上。然后，使用细钨针将每只眼睛从相关视叶上切开，并操纵每只眼睛到显微镜幻灯片的表面，使眼睛的基底表面与幻灯片相邻。然后，在样品上涂抹一个干净的盖玻片，然后用指甲油密封盖玻片。为了准备西方印迹分析的幼眼组织，首先解剖10至16只幼崽，如先前在两批中展示的，在PBS中相隔10至15分钟，辅以蛋白酶和磷酸酶抑制剂。在PBS加抑制剂的冰上九孔玻璃盘的单个孔中短暂冲洗分离眼脑复合物两次后，将所有眼脑复合物转移到400微升的冰冷PBS和抑制剂中，并放入干净的黑色解剖盘中。在干净的解剖显微镜下，使用坚固的钨针和细剃刀刀片从视叶上清洁切开每只眼睛，并使用润滑尖端将眼脑复合物转移到五微升的PBS加抑制剂中，放入冰上500微升微离心管中。然后，在管中加入5微升的西组织解液缓冲液和10微升的2倍浓缩蛋白样品缓冲液。处理用于RNA提取的眼脑复合物，戴上手套，清洁台面、显微镜、解剖工具，以及使用70%乙醇和RNase去污试剂的黑色解剖盘。在允许该区域干燥后，共解剖30至40只幼崽，分批分6至8次。每批眼脑复合物经过解剖后，将眼脑复合物从九井盘转移到400微升的新鲜、冰冷、无核酸的PBS中，用坚固的钨针和细剃刀刀片清洁切出视叶的每只眼睛。当一批眼睛全部被移除后，将它们转移到无菌的无RNase微离心管中，并在液氮中快速冷冻样品，然后再进入下一批。幼眼是一种易于接近的组织，是研究发育过程（包括驱动形态形成的过程）的极佳模型。在幼崽形成后约40小时，细胞的最终排列达到。这是一个理想的年龄，用于评估基因突变或改性基因表达的后果。例如，在凋亡的核心抑制剂Diap1的异位表达之后，与在控制视网膜中观察到的晶格细胞数量的增加可以进行比较。通过利用抗体激活死亡木塞-1或使用其他方法来检测垂死的细胞，也可以更直接地评估凋亡。西方眼酸的印迹分析可用于确定感兴趣的蛋白质的存在或相对表达，或比较不同发育时间点的蛋白质表达。值得注意的是，每基因型或实验条件解剖超过60只眼睛是隔离足够高质量RNA的最佳选择。最重要的是要记住的是，幼崽视网膜是非常脆弱的，在解剖过程中必须非常小心，以确保其完整性。

Research

JoVE Journal

Developmental Biology

Dissection of the Drosophila Pupal Retina for Immunohistochemistry, Western Analysis, and RNA Isolation

Large-scale Zebrafish Embryonic Heart Dissection for Transcriptional Analysis

大规模的斑马鱼胚胎的心脏解剖的转录分析

The zebrafish embryonic heart is composed of only a few hundred cells, representing only a small fraction of the entire embryo. Therefore, to prevent the ...

科研

发育生物学

Live-imaging of the Drosophila Pupal Eye

Live-imaging of the Drosophila Pupal Eye

实时成像<em&gt;果蝇</em&gt;蛹眼

Inherent processes of Drosophila pupal development can shift and distort the eye epithelium in ways that make individual cell behavior difficult to track ...

Isolation of Neural Stem/Progenitor Cells from the Periventricular Region of the Adult Rat and Human Spinal Cord

从脑室周围地区的成年大鼠和人类脊髓神经分离干/祖细胞

Adult rat and human spinal cord neural stem/progenitor cells (NSPCs) cultured in growth factor-enriched medium allows for the proliferation of ...

A Rapid and Efficient Method to Dissect Pupal Wings of Drosophila Suitable for Immunodetections or PCR Assays

A Rapid and Efficient Method to Dissect Pupal Wings of Drosophila Suitable for Immunodetections or PCR Assays

一种快速高效的解剖蛹翅的方法, 适用于 Immunodetections 或 PCR 检测

Wing development in Drosophila melanogaster is an ideal model for studying morphogenesis at tissue level. These appendages develop from a group of cells ...

Correlative Super-resolution and Electron Microscopy to Resolve Protein Localization in Zebrafish Retina

斑马鱼视网膜蛋白定位的相关超分辨和电镜观察

We present a method to investigate the subcellular protein localization in the larval zebrafish retina by combining super-resolution light microscopy and ...

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

成人果蝇脑内蘑菇体和感光神经元的解剖和荧光染色

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the ...

Dissection and Staining of Drosophila Pupal Ovaries

Dissection and Staining of Drosophila Pupal Ovaries

果蝇蛹卵巢的解剖与染色

Unlike adult Drosophila ovaries, pupal ovaries are relatively difficult to access and examine due to their small size, translucence, and encasing within a ...

Thawing, Culturing, and Cryopreserving Drosophila Cell Lines

Thawing, Culturing, and Cryopreserving Drosophila Cell Lines

解冻、培养和冷冻保存的嗜酸性粒细胞系

There are currently over 160 distinct Drosophila cell lines distributed by the Drosophila Genomics Resource Center (DGRC). With genome engineering, the ...

Imaging and Analysis of Tissue Orientation and Growth Dynamics in the Developing Drosophila Epithelia During Pupal Stages

Imaging and Analysis of Tissue Orientation and Growth Dynamics in the Developing Drosophila Epithelia During Pupal Stages

普帕尔阶段发育性皮瘤中组织定向和生长动力学的成像与分析

Within multicellular organisms, mature tissues and organs display high degrees of order in the spatial arrangements of their constituent cells. A ...

Preparation of Drosophila Larval and Pupal Testes for Analysis of Cell Division in Live, Intact Tissue

Preparation of Drosophila Larval and Pupal Testes for Analysis of Cell Division in Live, Intact Tissue

用于活、完整组织中细胞分部分析的果蝇拉瓦尔和普帕尔睾试验的制备

Experimental analysis of cells dividing in living, intact tissues and organs is essential to our understanding of how cell division integrates with ...