我们感谢名古屋大学的哉 Asai 为自由漂浮空肠的低温抗原提取提供了有益的建议。这项工作得到了 2012年 (JP25460493) 和 (B) 2017年 (JP17H04065) 从日本促进科学学会 (KAKENHI) 获得的援助资助, 一步赠款在 2014年 (AS251Z02522Q) 和在 2015年 (AS262Z00715Q) 从日本科技局 (JST), 和武田的前瞻研究赠款2014从武田科学基金会 (硕士)。

这里描述的所有方法都是由爱知人类服务中心发展研究研究所动物护理和使用委员会 (申请编号: M-03) 批准的。
1. 筹备工作
<ol><li>准备10升磷酸盐缓冲盐水 (PBS): 32.27 克 Na2HPO4· 12H2O, 4.5 克 NaH2PO4· 2H2O, 80.0 克氯化钠添加到超纯水中, 在室温 (RT) 的最后容积 10 l。</li>
	<li>准备200毫升的 pbs t: 200 毫升的 pbs 从步骤1.1 与100µL 聚氧乙烯 (10) 对辛醚到最后浓度 0.05% (卷: 卷)。存储在 RT。</li>
	<li>在佩戴手套和眼部保护的同时, 在10% 缓冲中性福尔马林溶液中准备50毫升15% 蔗糖: 7.5 克蔗糖添加到10% 缓冲中性福尔马林溶液 (材料表), 最终体积为50毫升。存储在 RT。 注意: 吸入和/或皮肤/眼睛接触甲醛在10% 缓冲中性福尔马林溶液可能是危险的。小心处理。</li>
	<li>准备1.5 毫升200单位/毫升罗丹明荧光染料共轭溶液: 罗丹明荧光染料共轭 (300 单位在1瓶, 冻干固体, 激发和发射波长: 581 毫微米和609毫微米分别) 悬浮在1.5 毫升甲醇.存储解决方案在黑暗中的-20 °c。</li>
	<li>准备5毫克/毫升 4 ', 6-diamidino 2-苯基吲哚, 盐酸盐 (DAPI) 库存溶液:10 毫克 DAPI 在2毫升的 n, n-甲基酰胺 (DMF)。存储解决方案在黑暗中的-20 °c。</li>
	<li>在 pbs 中制备5% 牛血清白蛋白 (bsa): 0.5 克 bsa, 10 毫升 pbs。存储在4摄氏度。</li>
	<li>使用一个钳移除18口径直针的斜面尖端, 并将夹紧端与一个在纵向方向上的守财奴捏在一起, 使液体流经针腔 (图 1A1)。 注意: 协议可以在这里暂停。</li>
</ol>2. 空肠的动物解剖和分离
<ol><li>小鼠灌注固定术<ol>
<li>戴上手套, 在通风良好的地方工作。</li>
		<li>准备一个100毫升的玻璃烧杯, 10% 缓冲中性福尔马林溶液, 外科工具 (剪刀, 镊子), 金属托盘, 6-0 尼龙切割缝合, 18 口径直针制备为 1.7, 22 口径翅针, 20 毫升注射器。</li>
		<li>将20毫升10% 缓冲中性福尔马林溶液从玻璃烧杯装入20毫升注射器, 并将22口径翅针连接到出口处。</li>
		<li>将1克明胶粉加入20毫升的 PBS (最终5% 明胶), 在50毫升离心管中制备液体明胶。浸泡在 RT 15 分钟后, 在50摄氏度的水浴中孵育15分钟, 不摇晃。</li>
		<li>简要地漩涡, 并且孵育在50°c 为另外15分钟完全地溶化明胶。把管子留在 RT 上, 直到使用。</li>
		<li>弄死一只成年小鼠颈椎脱位。</li>
		<li>将鼠标从步骤2.1.6 放在金属托盘上, 通过剑软骨在皮肤上做一个小切口, 使用手术工具。</li>
		<li>用双手扩大切口, 以暴露胸腔和腹部区域。</li>
		<li>打开腹膜以暴露隔膜, 然后打开两侧的隔膜。</li>
		<li>将胸笼沿两侧前腋线切开, 然后在胸腺的位置横截切口以暴露心脏。</li>
		<li>在右心房的耳廓上做一个小切口, 排出血液, 将22口径的翅针插入左心室的顶端。用10% 缓冲中性福尔马林溶液将柱塞灌注组织。</li>
		<li>在骨盆内暴露器官后, 从肛门切除直肠的末端, 通过切开肠系膜将肠道与身体分开。</li>
		<li>分离空肠, 从胃窦肛门侧切开4厘米的肠道。丢弃其余小肠的肛门半部分。</li>
		<li>将空肠夹在半空, 以方便冲洗工序。将20毫升10% 缓冲中性福尔马林溶液装入20毫升注射器, 并将步骤1.7 中准备的18口径直针连接到出口。</li>
		<li>从修剪的空肠一端注入10% 缓冲中性福尔马林溶液, 冲洗肠道内容, 修复肠道腔表面 (图 1A2)。</li>
		<li>按步骤2.1.15 中所述的注射 PBS 冲洗肠道腔。</li>
		<li>将液体明胶冲入肠道腔内, 如步骤2.1.15 中所述, 以液体明胶取代 PBS。</li>
		<li>用6-0 尼龙切口缝合缝合空肠的一端, 用液体明胶填充空肠, 并用缝合结扎 (图 1A3) 关闭相反端。</li>
		<li>添加四缝合节, 使香肠形状的空肠部分适合 cryomold 的郁闷部分 (图 1A4)。 注: 对于 cryomolds 与20毫米 x 25 毫米 x 5 毫米凹陷部分, a ~ 20 毫米香肠状空肠部分是可取的。</li>
		<li>将50毫升15% 蔗糖的组织浸泡在10% 个缓冲中性福尔马林溶液中, 隔夜在4摄氏度。 注: 这些步骤确保抗冻的蔗糖和蛋白质固定的甲醛的明胶和组织。福尔马林-糊明胶不熔化在 rt, 而非固定糊明胶在4°c 融化在 rt。</li>
	</ol>
</li>
</ol>3. 凝胶充填空肠组织冻结
<ol><li>通过在缝合节上切开空肠, 获得三个具有两端结扎的香肠状空肠 (图 1A4)。在 cryomold 中对香肠样件进行排列, 以添加嵌入化合物, 用于制备冷冻组织标本。</li>
	<li>用液氮冷却异戊烷中的 cryomold。存储 cryomolds 在-80 °c 注意: 协议可以在这里暂停。</li>
</ol>4. 游离漂浮 Cryosections 对一簇细胞免疫荧光的研究
<ol><li>Cryosectioning<ol>
<li>将恒温器室温度 (CT) 和目标温度 (OT) 设置为-22 摄氏度。将冰冻组织块放置在恒温器室的 cryomold 中, 至少15分钟。</li>
		<li>将3毫升 PBS 添加到35毫米培养皿中。</li>
		<li>从 cryomold 中取出冰冻的组织块, 用剃刀刀片将块切成两半, 露出空肠的横断面。将块的一半装在一个恰克 (恒温器适配器) 上, 用剃刀刀片截割平面。</li>
		<li>将明胶充填的空肠分成30µm 厚的部分。使用冷冻钳将节轻轻地转移到在步骤4.1.2 中准备的 35 mm 培养皿中 (图 2B, 右)。</li>
	</ol>
</li>
	<li>原发性抗体的应用<ol>
<li>在35毫米培养皿中, 每3次清洗自由浮动的部分5分钟, 每个与3毫升 PBS-T 与轻微震动在一个相互的振动筛 (大约36次/分钟)。</li>
		<li>准备抗原检索解决方案: 0.3 毫升抗原检索溶液浓缩 (材料表) 在2.7 毫升的超纯水。加入3毫升的抗原检索解决方案, 35 毫米培养皿中含有游离漂浮的部分。</li>
		<li>关闭盖子, 用一条乙烯胶带封住盘子和盖子之间的缝隙, 在50摄氏度的杂交孵化器中孵育3小时, 不摇晃。</li>
		<li>从孵化器中取出盘子, 在 RT 上冷却20分钟。取出乙烯基胶带后, 每段3次冲洗5分钟, 每次3毫升的 PBS 和轻微震动。</li>
		<li>吸入 PBS 并将5滴阻塞溶液 (材料表) 添加到各节上。在 RT 中孵育5分钟, 轻度晃动。</li>
		<li>准备主要抗体溶液: 495 µL 5% BSA 在 PBS 与5µL phospho-Y1798 girdin (pY1798) 抗体 (材料表)。将主抗体解的500µL 添加到各节 (不需要删除阻止解决方案)。</li>
		<li>把盘子放在一个湿润的孵化室里, 在4摄氏度的时候用轻微的震动孵化一夜。 注意: 夜间孵化可延长至三晚。</li>
	</ol>
</li>
	<li>二次抗体的应用<ol>
<li>在3毫升的 PBS 中, 用轻微的震动洗涤3次, 每节5分钟。</li>
		<li>准备稀释 DAPI 库存解决方案: 2 µL 的 DAPI 库存解决方案 (步骤 1.5), 998 µL 的 PBS。</li>
		<li>做二次抗体解决方案: 476 µL 5% BSA 在 PBS, 10.5 µL 稀释 DAPI 溶液, 12.5 µL 罗丹明荧光染料共轭股票溶液, 1 µL 山羊抗兔 IgG-荧光染料共轭 (波长: 励磁496毫微米, 发射520毫微米)。</li>
		<li>在对 PBS 进行吸吸后, 应用二次抗体溶液, 并在30分钟内以轻度晃动的方式在光屏蔽孵化室中孵育。</li>
		<li>用3毫升的 PBS 和轻微的震动, 每段3次冲洗5分钟。</li>
		<li>最后清洗后, 去除 pbs T, 并更换3毫升的 pbs 缺乏聚氧乙烯 (10) 对辛醚。把盘子转到立体显微镜上。</li>
		<li>将200µL 的 PBS 放在一个小水滴上, 并将一个空肠部分从盘子中转移到液滴上, 使用 P200 吸管尖。</li>
		<li>调整后, 在立体显微镜下的部分对准, 吸入所有其余的 PBS 周围的部分。</li>
		<li>添加20µL 的水上安装介质, 并在介质上放置 20 x 20 毫米盖玻片。</li>
		<li>立即用二甲苯为基础的安装介质密封盖玻片边缘。将幻灯片放在木制 mappe 上, 并允许以二甲苯为基础的安装介质在 RT 上凝固 2-3 小时。 注意: 协议可以在这里暂停。在以二甲苯为基础的安装介质固化后, 可以在 RT 的光屏蔽滑动盒中存储 2-3 周的幻灯片。</li>
	</ol>
</li>
</ol>5. 共焦显微术
<ol><li>将浸没油放在共焦显微镜的63X 目标上。将盖玻片向下滑动, 然后将幻灯片放在舞台上。</li>
	<li>将在激光波长405、488和 555 nm 获取的 TC 图像数字化, 并以 tiff 格式 (. tiff) 保存图像。 注: 选择适合于荧光染料的检测过滤器集 (即激发/发射最大值: 358/461 nm、490/525 和 590/617)。</li>
</ol>

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作者没有什么可透露的。

该协议的目的是让研究人员没有组织学经验, 以获得可靠的图像束细胞 (TCs)。该协议有三关键点: 1) 使用的网站和磷酸化状态特定的兔多克隆抗体抗人 girdin 磷酸化在酪氨酸 1798 (pY1798 抗体), 从一个特定的公司获得 (免疫生物实验室 = 公司 X);2) 空肠明胶充填;3) 低温抗原的提取。从广义上说, 磷酸化状态特异抗体可归类为修饰特异抗体, 用于识别特定类型的平移后修饰 (例如乙酰化、甲基化或磷酸化)。特定氨基酸残留物中的蛋白质。大森et 。X 公司联合生成 pY1798 抗体, 通过免疫磷酸化肽对兔进行接种, 并用固相色谱与 unphosphorylated 肽纯化生成的抗体. 在 Goto 方法910. pY1798 抗体通过体外磷酸化实验进行了密集的验证, 使用全长的人类 girdin 表达载体, 并在酪氨酸 17989中没有点突变。随后, 库加et发现, X 公司的 pY1798 抗体是肠道 TCs1的一个特异且敏感的标记。因此, 将 pY1798 抗体纳入 X 公司是本议定书的一个关键点。
明胶含有从动物组织中提取的胶原蛋白, 长期以来一直被用于用 cryosections13嵌入组织标本进行细胞化学研究。低熔点明胶, 这是糊在4摄氏度, 但在室温下融化, 开始应用, 以避免在嵌入14期间, 保持凝胶在液体状态所需的热量的不良影响。本协议采用明胶粉制成的低熔点明胶, gelatinizes 4 摄氏度, 并在 RT 中熔化, 以保持小鼠空肠横 cryosections 的形态。当这种明胶用于完全填充空肠时, 样品然后福尔马林固定, 以达到不可逆转的糊化。热诱导抗原检索是一种有效的方法, 用于暴露由含醛护12屏蔽的抗原。然而, 高温 (95-99 °c 为30分钟) 通常用于分析石蜡切片可能损害组织/明胶复合体的形态学。在这里, 我们使用低温抗原检索 (50 °c 为3小时), 允许抗原检索和保存组织形态学。
pY1798 抗体不仅能在小鼠空肠上标记 TCs, 而且还可以在多脏器 (胃、回肠、结肠、胆囊) 中贴上小鼠和人类的标签1。然而, 由于任何组织中未固定组织的 pY1798 信号会迅速降低, 此协议包括福尔马林注入空肠腔 (步骤 2.1.15,图 1A2)。
自由浮动 cryosections 的厚度有限制。虽然薄 cryosections 在显微镜的半透明性方面可能是有利的, 但薄层使这些部分的身体脆弱。相比之下, 厚 cryosections 是物理上坚固, 但有较低的抗体渗透性的部分。在本议定书中, 使用了30µm 厚的部分, 既物理上稳定和渗透抗体。虽然这种方法是为没有丰富的组织学经验的研究人员设计的, 如果该协议失败, pY1798/villin/DAPI 三重染色的石蜡切片类似于库加et 。可以执行1。石蜡切片在这里没有使用, 以避免与罗丹明染色的 F 肌动蛋白在石蜡切片的困难。
由于 girdin tyrosine-1798 附近的氨基酸序列的保护, pY1798 抗体可用于除鼠和人以外的其他物种中对 TCs 的染色。本协议中使用的明胶充填物也可应用于其它中空器官。事实上, 除了 pY1798 或 TC, 凝胶充填与低温抗原检索的结合可能有助于揭示众所周知的 "困难" 表位在老鼠肠道, 并因此, 可能是对许多研究者感兴趣。
girdin 的生物学作用及其磷酸化在 TCs 中的意义尚不清楚。然而, 林中的一项研究则是et 。提示酪氨酸磷酸化 girdin 与 F 肌动蛋白聚合的程度有关8。同时, 库加et 等发现, 致命剂量的凋亡诱导剂 (e. g, 顺铂, X 射线辐射) 引起的小鼠小肠 TCs 的相对频率大增加, 他们假设可能与从 enterocytes 到 TCs 的可能转换, 与微绒毛1中 girdin 的磷酸化同步。这种可能性今后还需要进一步调查。三组最近观察到寄生虫感染后 TC 频率迅速增加15,16,17。因此, 这种游离漂浮染色不仅可以用于分析内窥镜收集的人体肠道组织, 而且 TC 积累也有助于诊断寄生虫感染的人肠道。

簇状细胞 (TCs) 是胃肠上皮的微小分散成分, 其特征是顶端的塔夫茨和线轴形 somas1。虽然 TCs 在 1956年2中首次被描述, 但 tc 功能仍不清楚, 部分原因是缺少可靠的 TC 标记。
Enomoto et 。首先特点的肌动蛋白结合蛋白质 girdin3, 这是表达在神经系统, 以及在非神经组织, 如血管, 心脏瓣膜, 肌腱, 骨骼肌肉4。girdin 基因消融小鼠显示生长迟缓和多发性脑异常4,5,6。同时, 人类 girdin 的功能丧失突变与渐进性脑病、严重迟缓和早发性癫痫发作有关7。
在 2011年, 林et在酪氨酸激酶 (如 EGFR 和 Src8) 中发现了 girdin 的动态磷酸化。他们还表明, 磷酸化 girdin 是需要的肌动蛋白重塑, 以触发细胞迁移8。大森et . 根据 Goto 的协议, 在酪氨酸 1798 (pY1798 抗体) 上开发了针对人类 girdin 磷酸化的部位和磷酸化状态特异抗体, 并通过现场定向诱变验证了抗体承载全长 girdin910的表达式向量。在 2017年, 库加et 。报告说, pY1798 标签 TCs 具有高特异性和高灵敏度1。pY1798 抗体表现出优于先前的 TC 标记, 基于优良的染色性能, 揭示了整个细胞的形状, 包括特征 "一簇" 的食管腔尖端, 但 girdin 和磷 girdin 在 TC 的生物学作用函数仍然不清楚1。
本研究所描述的方法涉及免疫荧光染色, 一种组织学技术, 用于标记一个特定的蛋白质在组织中使用一个主要抗体对靶蛋白和荧光共轭二级抗体的主要抗体.这种方法的目的是让具有有限组织学经验的研究人员获得高质量的 TC 图像。该协议使用自由浮动 cryosections, 避免了对幻灯片安装 cryosections 或石蜡切片的需要。然而, 自由浮动的部分是脆弱的, 由于缺乏支持从玻璃滑动和切片厚度可以减少抗体通透性。该协议包括两种克服这些问题的方法: 1) 用明胶填充整个空肠腔, 保持游离漂浮切片在整个染色过程中的形态学, 2) 利用低温抗原检索强化 pY1798 信号。

<table>
	<tbody>
		<tr>
			<td>Slc:DDY 6-week-old female mice</td>
			<td>Chubu Kagaku Shizai</td>
			<td>Not applicable</td>
			<td></td>
		</tr>
		<tr>
			<td>Disodium hydrogenphosphate 12-water (Na2HPO412H2O)</td>
			<td>Wako</td>
			<td>196-02835</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium dihydroenphosphate Dihydrate (NaH2PO42H2O</td>
			<td>Wako</td>
			<td>199-02825</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride (NaCl)</td>
			<td>Wako</td>
			<td>199-10665</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100,&#160; Polyoxyethylene(10) octylphenyl ether</td>
			<td>Katayama Chemical</td>
			<td>Not applicable</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Wako</td>
			<td>190-00013</td>
			<td></td>
		</tr>
		<tr>
			<td>10% buffered neutral formalin solution</td>
			<td>Muto Chemical</td>
			<td>20215</td>
			<td>Step 1.3.</td>
		</tr>
		<tr>
			<td>Phalloidin-fluorescent dye conjugate, Alexa Fluor 594 phalloidin</td>
			<td>ThermoFisher Scientific</td>
			<td>A12381</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Nacalai Tesque</td>
			<td>21915-35</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI (4&#39;,6-diamidino-2-phenylindole, dihydrochloride)</td>
			<td>ThermoFisher Scientific</td>
			<td>D1306</td>
			<td></td>
		</tr>
		<tr>
			<td>N.N-dimethylformamide (DMF)</td>
			<td>Nacalai Tesque</td>
			<td>13015-75</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Sigma</td>
			<td>A9647-10G</td>
			<td></td>
		</tr>
		<tr>
			<td>18-gauge stainless steel straight needle</td>
			<td>Terumo</td>
			<td>NN-1838R</td>
			<td></td>
		</tr>
		<tr>
			<td>U.S.P. 6-0, 60 cm nylon cut suture, Crownjun</td>
			<td>Kono Seisakusho</td>
			<td>Not applicable</td>
			<td></td>
		</tr>
		<tr>
			<td>22-gauge winged needle</td>
			<td>Terumo</td>
			<td>SV-22DLK</td>
			<td></td>
		</tr>
		<tr>
			<td>20 mL TERUMO syringe</td>
			<td>Terumo</td>
			<td>SS-20ES</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL centrifuge tube</td>
			<td>TPP</td>
			<td>91050</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL Iwaki centrifuge tubes</td>
			<td>Iwaki</td>
			<td>2325-015</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL micro test tube</td>
			<td>Star</td>
			<td>RSV-MTT1.5</td>
			<td></td>
		</tr>
		<tr>
			<td>Gelatin powder, Gelare-blanc</td>
			<td>Nitta Biolab</td>
			<td>2809</td>
			<td></td>
		</tr>
		<tr>
			<td>Embedding compound for frozen tissue specimen, O.C.T. Compound 118 mL 12 piece</td>
			<td>Sakura FineTech</td>
			<td>4583</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryomold, CRYO DISH No. 3, 125 piece, well size 20 x 25 x 5 (mm)</td>
			<td>Shoei Work&#39;s</td>
			<td>1101-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopentane</td>
			<td>Nacalai Tesque</td>
			<td>26404-75</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon cell culture dish 35 x 10mm Easy-Grip</td>
			<td>Corning</td>
			<td>353001</td>
			<td></td>
		</tr>
		<tr>
			<td>Antigen retrieval solution concentrate, Target Retrieval Solution pH 9 10x</td>
			<td>Dako</td>
			<td>S2367</td>
			<td>Antigen retrieval solution concentrate in step 4.2.2.</td>
		</tr>
		<tr>
			<td>Protein Block</td>
			<td>Dako</td>
			<td>X0909</td>
			<td>Blocking solution in 4.2.5.</td>
		</tr>
		<tr>
			<td>Phospho-Y1798 girdin (pY1798) antibodies</td>
			<td>Immuno-Biological Laboratories (IBL) = Company X</td>
			<td>28143</td>
			<td>Phospho-Y1798 girdin antibodies in step 4.2.6.</td>
		</tr>
		<tr>
			<td>Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488</td>
			<td>ThermoFisher Scientific</td>
			<td>A11034</td>
			<td></td>
		</tr>
		<tr>
			<td>Aquous mounting media, Prolong Gold Antifade Mountant</td>
			<td>ThermoFisher Scientific</td>
			<td>P36930</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslips, Matsunami Micro Cover Glass 22 x 22 mm 100 pcs Thickness No.1</td>
			<td>Matsunami Glass</td>
			<td>C022221</td>
			<td></td>
		</tr>
		<tr>
			<td>Entellan New, xylene-based mounting media for microscopy</td>
			<td>Merck Millipore</td>
			<td>107961</td>
			<td></td>
		</tr>
		<tr>
			<td>MAS-coated slide glass white</td>
			<td>Matsunami Glass</td>
			<td>MI-MAS-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Wooden Mappe KO-type</td>
			<td>Shoei Work&#39;s</td>
			<td>99-40007</td>
			<td></td>
		</tr>
		<tr>
			<td>Immersion Oil 518 F Fluorescence Free 20 ml</td>
			<td>Zeiss</td>
			<td>444960</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipettes, Pipetman P (P2, P20, P200, P1000)</td>
			<td>Gilson</td>
			<td>F144801, F123600, F123601, F123602</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrapure water production system</td>
			<td>Advantec</td>
			<td>GS-590</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic glove, Star Nitrile Glove</td>
			<td>Star</td>
			<td>RSU-NGVM</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryostat</td>
			<td>Leica Microsystems</td>
			<td>CM1950</td>
			<td></td>
		</tr>
		<tr>
			<td>Deep freezer, SANYO Ultra Low</td>
			<td>Sanyo</td>
			<td>MDF-382</td>
			<td></td>
		</tr>
		<tr>
			<td>Showcase refrigerator</td>
			<td>Nihon Freezer</td>
			<td>NC-ME50EC</td>
			<td></td>
		</tr>
		<tr>
			<td>Water bath, Thermominder SDminiN (to dissolve gelatin at 50 C)</td>
			<td>Taitec</td>
			<td>0068750-000</td>
			<td></td>
		</tr>
		<tr>
			<td>Hybridization incubator (for antigen retrieval at 50 C)</td>
			<td>Taitec</td>
			<td>HB-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubation chamber for immunostaining</td>
			<td>Cosmo Bio</td>
			<td>10DO</td>
			<td></td>
		</tr>
		<tr>
			<td>Reciprocal shaker for immunohistochemistry (for room temperature)</td>
			<td>Taitec</td>
			<td>NR-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Reciprocal shaker for immunohistochemistry (for 4 C)</td>
			<td>Tokyo Rika Kikai</td>
			<td>MMS-3010</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereoscopic microscope (for tissue handling)</td>
			<td>Olympus</td>
			<td>SZ61</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereoscopic microscope (for Figure 1B)</td>
			<td>Leica Microsystems</td>
			<td>M165FC</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence microscope (for Figure 3)</td>
			<td>Nikon</td>
			<td>E800</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal laser-scanning microscope</td>
			<td>Zeiss</td>
			<td>LSM700</td>
			<td></td>
		</tr>
	</tbody>
</table>

use of anti-phospho-girdin antibodies to visualize intestinal tuft cells in free-floating mouse jejunum cryosections

肌动蛋白结合蛋白质 girdin 是一种胞浆蛋白, 这是需要的肌动蛋白重塑, 以触发细胞迁移的各种组织。Girdin 是由受体和非受体酪氨酸激酶在酪氨酸1798磷酸化。大森et . 在 tyrosine-1798 (pY1798) 上开发了针对人类 girdin 的站点和磷酸化状态特异抗体, 专门与磷酸化 tyrosine-1798, 而不是 unphosphorylated tyrosine-1798。pY1798 抗体已被用来专门标记在哺乳动物胃肠组织中存在的一簇细胞 (TCs), 但这些细胞的功能是不清楚的。该协议允许使用 pY1798 抗体和免疫荧光技术在空肠中对 TCs 进行健壮的可视化。为了确保成功和简单的 TC 可视化, 该协议包括两种组织学技术: 生产游离漂浮 cryosections 从明胶充填空肠组织, 低温抗原检索50°c 3 小时. 充填空肠在整个染色过程中, 明胶保持自由浮动剖面的形状, 而低温抗原的提取则确保了 tcs 的鲁棒性信号. 该协议的成功使用导致 pY1798 染色从绒毛尖端分布到地穴.染色 TCs 有一个线轴状的躯体和荧光信号凝结在食管腔尖端, 这对应于突出的 ' 一簇。罗丹明染色 colocalized 与 pY1798-positive TCs 在加厚的画笔边界, 并对应于支根质量延伸从 TC 丛。本协议可用于检查用胃肠内窥镜采集的人体活检标本中的 TCs。此外, 最近据报道, TCs 在小鼠的寄生虫感染后积聚, 这表明该议定书可能对人类肠道寄生虫感染的诊断有应用价值。

空肠明胶充填
用明胶充填小鼠空肠的典型方法 (图 1A), 如明胶充填的好处 (图 1B)。简而言之, 18 尺直针的斜面尖端被移除以防止刺穿肠道壁 (图 1A1)。凝胶充填是使用10% 缓冲中性福尔马林溶液注入从修剪空肠部分的一端冲洗肠道和修复肠道腔表面 (图 1A2), 然后再填充液体明胶( 图 1A3)。产生的香肠样件 (图 1A4) 被嵌入、冻结和横向切片成恒温器中的30µm 厚切片。在没有明胶充填的情况下, 空肠部分趋于扭结, 使绒毛向后摆动 (图 1B, 左)。凝胶充填保留了圆形圆盘形状的部分, 并保持绒毛的直立定位 (图 1B, 右)。这些图像清楚地说明了明胶充填对保留游离空肠剖面形态的益处。
肠束细胞的成功影像学研究
pY1798 抗体可用于可变染色技术, 包括斑点印迹, 西方印迹, 石蜡切片染色, 解冻安装 cryosection 染色, 或自由浮动 cryosection 染色1,9。在本协议中, 我们重点研究了自由浮动 cryosections 在小鼠空肠荧光染色中的应用 pY1798 抗体, 罗丹明和 DAPI 染色, 以证明 tcs1的结构特征。一般而言, TCs 是分散在大约一个 TC/100 上皮细胞从绒毛尖到墓穴1。代表的结果表明, pY1798 重现性的整个 TCs, 包括膜, 细胞质的线轴状的躯体, 和强烈染色的食管腔尖端, 其中鲁棒信号凝结对应的突出 ' 一簇 'TC 1 (图 2A-2B)。同时, 罗丹明是一个 phallotoxin, 一家有毒的笼一类具有从蘑菇鹅蕈, 并具有很高的亲和力, 丝状肌动蛋白 (F 肌动蛋白), 这是存在于微绒毛, 形成肠道刷边界11. 罗丹明重现性和突出标记与从一束1扩展的大量根相对应的加厚画笔边框.因此, pY1798 信号 (线轴形的躯体, 信号凝结在食管腔尖端) 的一致的共同定位与显著加厚的罗丹明正刷边界表明, 该协议成功地识别 TCs, 无论无论它们位于绒毛(图 2A)还是在隐窝(图 2B)中。
低温抗原提取对游离漂浮切片的染色效果
热基抗原检索 95-99 °c 广泛用于分析石蜡切片和滑动安装 cryosections。但是, 将此方法应用于自由浮动节而不造成损坏是很困难的, 因为这些节通常比幻灯片12支持的幻灯片安装的部分更脆弱。由于低温抗原检索 (50 °c 为3小时) 不影响自由漂浮空肠剖面的形态学在初步实验中, 我们评估了抗原检索的客观效果在盲试验中, 这统计证实了低温抗原检索的有效性(图 3)。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57475/57475fig1.jpg"> 图 1: cryosections 的形态保存用空肠凝胶充填 (A)使用明胶在小鼠空肠腔内充填过程的照片。(A1)18尺直针的斜面尖端被移除, 以避免刺穿肠道壁。(A2)将缓冲中性福尔马林溶液 (10%) 注射在被修剪的空肠一端, 冲洗肠道内容, 修复肠腔表面。(A3)用6-0 尼龙缝合法 (黑色箭头) 填充液体明胶和两端结扎的截空肠(黑箭)。(A4)四更多缝合结(白色箭头)放置在预先存在的缝合结之间(黑箭头)产生了三个较短的片断。该组织将被分离成三香肠样件在两个指定的位置(白色箭头)。(B)明胶充填对自由漂浮 cryosection 形态的有益影响。如果没有凝胶充填, 切片倾向于扭结, 使绒毛容易向后摆动(左);与明胶充填, 30 µm 厚的部分保持一个圆盘形状和绒毛的直立位置保留(右)。图像使用 Nomarski 差分干涉对比拍照。刻度条, 1 毫米<a href="//ecsource.jove.com/files/ftp_upload/57475/57475fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57475/57475fig2.jpg"> 图 2: 小鼠小肠束细胞的代表性荧光图像 在绒毛(a)或隐窝(B)中的 TCs 的共焦荧光图像, 在酪氨酸1798上染色的 girdin 磷酸化的自由浮动小鼠空肠部分 (pY1798, 绿色, 最佳的励磁/发射波长490/525 毫微米), 罗丹明 (红色, 590/617 毫微米), 46-diamidino-2-苯基吲哚 (DAPI, 蓝色, 358/461 毫微米)。在低放大率图像 (缩放条, 50 µm) 中, 白色框所包围的区域在右侧 (缩放条、10µm) 展开。pY1798 抗体重现性染色 TCs, 无论位置 (在绒毛(a), 或在墓穴(B)), 染色目前在食管腔尖端 (箭头), 膜, 和细胞质的线轴状 TC 躯体。罗丹明染色 (箭头) 中明显加厚的画笔边框是 TCs 的另一个独特标志.<a href="//ecsource.jove.com/files/ftp_upload/57475/57475fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57475/57475fig3.jpg"> 图 3: 低温抗原检索增强 pY1798 免疫荧光 比较两组实验程序, 确定低温抗原检索的有效性。自由漂浮的空肠部分 (30 µm 厚, n = 13) 从一个单一的冰冻块被划分成两组, 每个组的部分被染色后, 完整的协议或同一协议缺乏低温抗原检索 (步骤 4.2.2)。染色后, 各切片被装在带有组号的单独幻灯片上, 每张幻灯片上的标签都用屏蔽胶带覆盖。所有的幻灯片被洗牌, 贴上新的数字在磁带上, 并观察到荧光显微镜下通过 FITC 过滤器。在每个组中平均可见 pY1798-positive TCs 的总计数, 并显示在条形图 (平均标准偏差)。对两组之间的平均计数进行了双尾 t 检验。P 值在0.05 以下被认为有统计学意义。低温抗原的提取大大提高了 pY1798-immunofluorescence 的有效性。<a href="//ecsource.jove.com/files/ftp_upload/57475/57475fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Use of Anti-phospho-girdin Antibodies to Visualize Intestinal Tuft Cells in Free-Floating Mouse Jejunum Cryosections at JoVE.com

Use of Anti-phospho-girdin Antibodies to Visualize Intestinal Tuft Cells in Free-Floating Mouse Jejunum Cryosections

抗磷 girdin 抗体在自由漂浮小鼠空肠 Cryosections 小肠簇细胞中的应用

库加et . 发现在酪氨酸 1798 (pY1798) 中, 抗肌动蛋白结合蛋白 girdin 磷酸化的特定抗体可用于标记一簇细胞 (TCs)。该协议允许使用 pY1798 抗体的自由漂浮空肠 cryosections 免疫荧光染色对 TCs 进行鲁棒可视化。

The actin binding protein girdin is a cytosolic protein that is required for actin remodeling to trigger cell migration in various tissues. Girdin is ...

use-anti-phospho-girdin-antibodies-to-visualize-intestinal-tuft-cells

Research

JoVE Journal

Biology

11.3K 次观看.  Aichi Human Service Center. 库加et . 发现在酪氨酸 1798 (pY1798) 中, 抗肌动蛋白结合蛋白 girdin 磷酸化的特定抗体可用于标记一簇细胞 (TCs)。该协议允许使用 pY1798 抗体的自由漂浮空肠 cryosections 免疫荧光染色对 TCs 进行鲁棒可视化。

视频: Use of Anti-phospho-girdin Antibodies to Visualize Intestinal Tuft Cells in Free-Floating Mouse Jejunum Cryosections

Catheterization of Intestinal Loops in Ruminants

The intestine is a complex structure that is involved not only in absorption of nutrients, but also acts as a barrier between the individual and the outside world. As such, the intestine plays a pivotal role in immunosurveillance and protection from enteric pathogens. Investigating intestinal physiology and immunology commonly employs 'intestinal loops' as an experimental model. The majority of these loop models are non-recovery surgical procedures that study short-term (&lt;24 hr) changes in the intestine (1-3). We previously created a recovery intestinal loop model to specifically measure long-term (&lt;6 mo) immunological changes in the intestine of sheep following exposure to vaccines, adjuvants, and viruses (4). This procedure localized treatments to a specific 'loop', allowing us to sample this area of the intestine. A significant drawback of this method is the single window of opportunity to administer treatments (i.e. at the time of surgery). Furthermore, samples of both the intestinal mucosa and luminal contents can only be taken at the termination of the project. Other salient limitations of the above model are that the surgical manipulation and requisite post-operative measures (e.g. administration of antibiotics and analgesics) can directly affect the treatment itself and/or alter immune function, thereby confounding results. Therefore, we modified our intestinal loop model by inserting long-term catheters into the loops. Sheep recover fully from the procedure, and are unaffected by the exteriorized catheters. Notably, the establishment of catheters in loops allows us to introduce multiple treatments over an extended interval, following recovery from surgery and clearance of drugs administered during surgery and the post-operative period.

We describe a novel surgical method for catheterizing 'intestinal loops' within the ileum of sheep. Once animals have recovered from surgery and have cleared antibiotics and analgesics, multiple treatments can be deposited directly in loops via the catheters.

导尿在反刍动物的肠袢

The intestine is a complex structure that is involved not only in absorption of nutrients, but also acts as a barrier between the individual and the ...

科研

生物学

A Technique to Simultaneously Visualize Virus-Specific CD8+ T Cells and Virus-Infected Cells In situ

The numbers and locations of virus-specific CD8+ T cells relative to the numbers and locations of their infected cell targets is thought to be critical in determining outcomes that range from clearance to chronic persistent infections. We describe here a method for assessing the spatial and quantitative relationships between immune effector (E) virus-specific CD8+ T cells and infected targets (T) that combines in situ tetramer (IST) staining to detect virus-specific CD8+ T cells and in situ hybridization (ISH) to detect viral-RNA+ cells in the tissues where the battle between immune defenses and infection takes place. The combination of IST staining and ISH, abbreviated ISTH, enables visualization and mapping of the locations of immune effector cells and targets, and facile determination of E:T ratios.  These parameters in turn can then be used to determine the relationships between spatial proximity, and the timing and magnitude of the immune response that predict outcomes in early infection.

A Technique to Simultaneously Visualize Virus-Specific CD8+ T Cells and Virus-Infected Cells In situ

A technique combining in situ tetramer staining and in situ hybridization (ISTH) enables visualization, mapping and analysis of the spatial proximity of virus-specific CD8+ T cells to their virus-infected targets, and determination of the quantitative relationships between these immune effectors and targets to infection outcomes.

一种技术，同时可视化病毒特异性CD8 + T细胞和病毒感染的细胞原位</em

The numbers and locations of virus-specific CD8+ T cells relative to the numbers and locations of their infected cell targets is thought to be critical in ...

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Human cancer and response to therapy is better represented in orthotopic animal models. This paper describes the development of an orthotopic mouse model of ovarian cancer, treatment of cancer via oral delivery of drugs, and monitoring of tumor cell behavior in response to drug treatment in real time using in vivo imaging system. In this orthotopic model, ovarian tumor cells expressing luciferase are applied topically by injecting them directly into the mouse bursa where each ovary is enclosed. Upon injection of D-luciferin, a substrate of firefly luciferase, luciferase-expressing cells generate bioluminescence signals. This signal is detected by the in vivo imaging system and allows for a non-invasive means of monitoring tumor growth, distribution, and regression in individual animals. Drug administration via oral gavage allows for a maximum dosing volume of 10 mL/kg body weight to be delivered directly to the stomach and closely resembles delivery of drugs in clinical treatments. Therefore, techniques described here, development of an orthotopic mouse model of ovarian cancer, oral delivery of drugs, and in vivo imaging, are useful for better understanding of human ovarian cancer and treatment and will improve targeting this disease.

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy. A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells. Treatment is administered via oral gavage. Tumor growth is monitored by in vivo imaging system.

活体成像和治疗卵巢癌的原位小鼠模型

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

Analysis of Pluripotent Stem Cells by using Cryosections of Embryoid Bodies

Embryonic stem (ES) cells are pluripotent cells derived from the inner cell mass of blastocyst-stage early mammalian embryos 1. A crucial stage in the differentiation of ES cells is the formation of embryoid bodies (EBs) aggregates 2, 3. EB formation is based on spontaneous aggregation when ES cells are cultured in non adherent plates. Three-dimensional EB recapitulates many aspects of early mammalian embryogenesis and differentiate into the three germ layers: ectoderm, mesoderm and endoderm 4.
Immunofluorescence and in situ hybridization are widely used techniques for the detection of target proteins and mRNA present in cells of a tissue section 5, 6, 7. Here we present a simple technique to generate high quality cryosections of embryoid bodies. This approach relies on the spatial orientation of EB embedding in OCT followed by the cryosection technique. The resulting sections can be subjected to a wide variety of analytical procedures in order to characterize populations of cells containing certain proteins, RNA or DNA. In this sense, the preparation of EB cryosections (10&mu;m) are essential tools for histology staining analysis (e.g. Hematoxilin and Eosin, DAPI), immunofluorescence (e.g. Oct4, nestin) or in situ hybridization. This technique can also help to understand aspects of embryogenesis with regards to the maintenance of the tri-dimensional spherical structure of EBs.

Pluripotent stem cells growing in suspension differentiate into embryoid bodies (EBs). Here we demonstrate how to obtain high quality EB cryosections useful for studying cellular and molecular aspects of embryogenesis, while preserving their organization as aggregates.

使用胚体冰冻切片分析的多能干细胞

Embryonic stem (ES) cells are pluripotent cells derived from the inner cell mass of blastocyst-stage early mammalian embryos 1. A crucial stage in the ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

诱导多能干细胞来源于小鼠的生成

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

Use of LysoTracker to Detect Programmed Cell Death in Embryos and Differentiating Embryonic Stem Cells

Programmed cell death (PCD) occurs in adults to maintain normal tissue homeostasis and during embryological development to shape tissues and organs1,2,6,7. During development, toxic chemicals or genetic alterations can cause an increase in PCD or change PCD patterns resulting in developmental abnormalities and birth defects3-5. To understand the etiology of these defects, the study of embryos can be complemented with in vitro assays that use differentiating embryonic stem (ES) cells.
Apoptosis is a well-studied form of PCD that involves both intrinsic and extrinsic signaling to activate the caspase enzyme cascade. Characteristic cell changes include membrane blebbing, nuclear shrinking, and DNA fragmentation. Other forms of PCD do not involve caspase activation and may be the end-result of prolonged autophagy. Regardless of the PCD pathway, dying cells need to be removed. In adults, the immune cells perform this function, while in embryos, where the immune system has not yet developed, removal occurs by an alternative mechanism. This mechanism involves neighboring cells (called "non-professional phagocytes") taking on a phagocytic role-they recognize the 'eat me' signal on the surface of the dying cell and engulf it8-10. After engulfment, the debris is brought to the lysosome for degradation. Thus regardless of PCD mechanism, an increase in lysosomal activity can be correlated with increased cell death.
To study PCD, a simple assay to visualize lysosomes in thick tissues and multilayer differentiating cultures can be useful. LysoTracker dye is a highly soluble small molecule that is retained in acidic subcellular compartments such as the lysosome11-13. The dye is taken up by diffusion and through the circulation. Since penetration is not a hindrance, visualization of PCD in thick tissues and multi-layer cultures is possible12,13. In contrast, TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) analysis14, is limited to small samples, histological sections, and monolayer cultures because the procedure requires the entry/permeability of a terminal transferase.
In contrast to Aniline blue, which diffuses and is dissolved by solvents, LysoTracker Red DND-99 is fixable, bright, and stable. Staining can be visualized with standard fluorescent or confocal microscopy in whole-mount or section using aqueous or solvent-based mounting media12,13. Here we describe protocols using this dye to look at PCD in normal and sonic hedgehog null mouse embryos. In addition, we demonstrate analysis of PCD in differentiating ES cell cultures and present a simple quantification method. In summary, LysoTracker staining can be a great complement to other methods of detecting PCD.

We present a simple protocol to visualize regions of programmed cell death (PCD) in mouse embryos and differentiating embryonic stem (ES) cell cultures using a highly soluble dye called LysoTracker.

使用LysoTracker检测及鉴别胚胎干细胞在胚胎细胞程序性死亡

Programmed cell death (PCD) occurs in adults to maintain normal tissue homeostasis and during embryological development to shape tissues and ...

Use of Time Lapse Microscopy to Visualize Anoxia-induced Suspended Animation in C. elegans Embryos

Caenorhabdits elegans has been used extensively in the study of stress resistance, which is facilitated by the transparency of the adult and embryo stages as well as by the availability of genetic mutants and transgenic strains expressing a myriad of fusion proteins1-4. In addition, dynamic processes such as cell division can be viewed using fluorescently labeled reporter proteins. The study of mitosis can be facilitated through the use of time-lapse experiments in various systems including intact organisms; thus the early C. elegans embryo is well suited for this study. Presented here is a technique by which in vivo imaging of sub-cellular structures in response to anoxic (99.999% N2; &lt;2 ppm O2) stress is possible using a simple gas flow through setup on a high-powered microscope. A microincubation chamber is used in conjunction with nitrogen gas flow through and a spinning disc confocal microscope to create a controlled environment in which animals can be imaged in vivo. Using GFP-tagged gamma tubulin and histone, the dynamics and arrest of cell division can be monitored before, during and after exposure to an oxygen-deprived environment. The results of this technique are high resolution, detailed videos and images of cellular structures within blastomeres of embryos exposed to oxygen deprivation.

Use of Time Lapse Microscopy to Visualize Anoxia-induced Suspended Animation in C. elegans Embryos

Described here is an in vivo technique to image sub-cellular structures in animals exposed to anoxia using a gas flow through microincubation chamber in conjunction with a spinning disc confocal microscope. This method is straightforward and flexible enough to suit a variety of experimental parameters and model systems.

使用时间的推移显微镜可视化缺氧诱导假死<em&gt; C.线虫</em&gt;胚胎

Caenorhabdits elegans has been used extensively in the study of stress resistance, which is facilitated by the transparency of the adult and embryo stages ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

诱导上皮和分析间质转化

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid decline in renal function and development of the clinical syndrome known as acute kidney injury (AKI). Pharmacological agents used to treat medical circumstances ranging from bacterial infection to cancer, when administered individually or in combination with other drugs, can initiate AKI. Zebrafish are a useful animal model to study the chemical effects on renal function in vivo, as they form an embryonic kidney comprised of nephron functional units that are conserved with higher vertebrates, including humans. Further, zebrafish can be utilized to perform genetic and chemical screens, which provide opportunities to elucidate the cellular and molecular facets of AKI and develop therapeutic strategies such as the identification of nephroprotective molecules. Here, we demonstrate how microinjection into the zebrafish embryo can be utilized as a paradigm for nephrotoxin studies.

Renal injuries incurred from nephrotoxins, which include drugs ranging from antibiotics to chemotherapeutics, can result in complex disorders whose pathogenesis remains incompletely understood. This protocol demonstrates how zebrafish can be used for disease modeling of these conditions, which can be applied to the identification of renoprotective measures.

肾毒素显微注射斑马鱼到模型急性肾损伤

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

We present a software solution for semi-automated tracking of relative protein concentration along the length of dynamic cellular protrusions.

软件辅助跟踪动态细胞突变中蛋白质浓度的图形用户界面

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...