作者感谢保罗托马斯显微镜的意见和帮助。这里所涉及的这项工作得到了 BBSRC (格兰特号) 的支持。BB/J009040/1 到 M.M.M. 和 t.w)。

这里描述的所有方法都是根据东英吉利大学的机构许可准则进行的。
1. 肠道组织的分离
<ol>
	<li>免疫标记结肠棺的分离 (参见图 1, 示意图)
	<ol>
		<li>安乐的鼠标 (使用 CO2窒息) 和删除冒号 (开始在盲肠和提取尾) 与解剖剪刀和镊子14。</li>
		<li>使用带有橡胶灯泡的玻璃吸管将结肠的含量与磷酸盐缓冲盐水 (PBS) 齐平。PBS: 氯化钠 (8.0 克/升)、氯化钾 (0.2 克/升)、磷酸氢二钠 (1.15 克/升) 和磷酸盐 (0.2 克/升), pH 7.3。</li>
		<li>使用金属棒 (2.4 毫米直径) 或玻璃吸管的末端, 握住结肠管的一端, 同时轻轻地将组织移到棒/吸管上, 从而外翻结肠组织, 使上皮细胞现在在外面。 注意: 如果这是不可能的然后打开冒号与剪刀和切片成5毫米片断。</li>
		<li>将外翻结肠或片转移到50毫升的锥形管中, 其中含有40毫升的 PBS。</li>
		<li>倒置管多次, 以进一步去除肠道内容, 粘液,等</li>
		<li>在 PBS 中将结肠/片转移到40毫升的3毫米 EDTA, 室温下孵育15分钟。 注: 用 PBS 稀释0.5 米 EDTA pH 8.0 的股票。</li>
		<li>摇动去除粘液, 将结肠/片转移到50毫升的锥形管中, 在 PBS 中含有新鲜40毫升的3毫米 EDTA。在 RT 处孵育35分钟。</li>
		<li>将结肠/片转移到10毫升 PBS 在50毫升锥形管和动摇大力三十年代释放棺 (地穴分数)。</li>
		<li>将结肠/片从试管中取出, 放回3毫米的 EDTA 中, 以防需要进一步隔离。离心剩余的墓穴分数在 300 x g 为 5 min。</li>
		<li>去除5毫升的上清和重的地穴颗粒在余下的5毫升的体积。</li>
		<li>观察下显微镜与变焦 (50-100X 放大), 以检查是否存在结肠棺。 注意: 如果不存在棺, 则在 PBS 中孵育3毫米 EDTA 中的结肠/片断, 再进行30分钟, 然后重复步骤 1.1.8-1.1.11。</li>
		<li>离心的地穴分数在 300 x g 为5分钟。</li>
		<li>取出所有上清液获得一个地穴颗粒并立即进行固定 (3 节)。</li>
	</ol>
	</li>
	<li>免疫标记小肠棺和绒毛的分离 (图 2)
	<ol>
		<li>安乐小鼠 (使用 CO2窒息), 用解剖剪刀和镊子14将小肠 (近端十二指肠到末端回肠) 取出。 注意: 要查看小肠的不同部分, 然后在这个阶段分开和治疗分开。有关流程图和计时, 请参见图 1和表 1 。</li>
		<li>使用带有橡胶灯泡的玻璃吸管, 用 PBS 冲洗小肠的内容。</li>
		<li>切开小肠与解剖剪刀和地方在15毫升 PBS 在一个培养皿。</li>
		<li>在 PBS 中轻轻冲洗肠道, 去除腔的内容, 同时不损害粘膜表面。将组织转移到新鲜的培养皿中并重复 PBS 洗涤。</li>
		<li>将小肠切成3-5 毫米, 转移到100毫米的培养皿中, 在 pbs 中含有15毫升的30毫米 edta, 在 RT 中孵育5分钟. 交替孵育3毫米 edta 在 pbs 中为30分钟。</li>
		<li>分数 1 (绒毛隔离): 将肠道碎片转化为50毫升锥形管中的 PBS 10 毫升, 用力摇动, 十年代, 倒入 100 mm 培养皿中。检查显微镜下孤立绒毛的分数。在这个阶段, 大多数绒毛应该是存在的, 但这可以改变之间的隔离。 注意: 为了防止孤立的绒毛/棺粘附在塑料上, 培养皿和收集管应预胎儿牛血清 (FBS)。将 FBS 倒入盘子和/或管子中, 立即将其除去。有关每个分数的计时指南, 请参见表 1 。</li>
		<li>将肠片移植到 PBS 的30毫米 EDTA 中, 孵育5分钟。在这个孵化期间, 收集分数1在15毫升圆锥管 (涂与 FBS) 和离心机在 300 x g 为5分钟。</li>
		<li>分数 2: 将肠片转移到50毫升的锥形管中, 其中含有10毫升 PBS, 二十年代剧烈摇动, 然后倒入 100 mm 培养皿中。检查显微镜下的分数。典型地, 在这个阶段绒毛和棺混合的文化存在 (图 2A)。</li>
		<li>将肠片移植到 PBS 的30毫米 EDTA 中, 再孵育5分钟。在此孵化期间, 颗粒分数2在15毫升锥形管 (涂与 FBS) 在 300 x g 为 5 min. 从馏分1和2中除去上清, 不干扰颗粒, 并立即进行固定 (步骤 3)。</li>
		<li>分数 3 (地穴隔离): 将肠道碎片转换为10毫升的 PBS 在50毫升管, 动摇二十年代, 并倒入100毫米培养皿。检查显微镜下的分数。在此阶段, 分数将主要包含棺 (图 2B)。</li>
		<li>分数 4: 将肠片转移到 PBS 的10毫升, 摇动二十年代, 倒入100毫米的培养皿中。检查显微镜下的分数。在这个阶段仅棺应该存在。</li>
		<li>分数 5: 将肠片转移到 PBS 的10毫升, 摇动二十年代, 倒入100毫米的培养皿中。检查显微镜下的分数。通常, 在这个阶段, 很少棺被提取。如果提取了多个棺, 则继续使用 6th分数。 注意: 如果一个纯粹的地穴人口是需要的 (例如, 为化世代), 然后使用70µm 细胞过滤器去除所有原封绒毛从地窖被丰富的分数。</li>
		<li>收集分数 3-5 在单独的15毫升圆锥管和离心机在 300 x g 为5分钟。</li>
		<li>检查分数 3-5 的颗粒, 去除清, 不干扰颗粒, 立即进行固定 (3 节)。</li>
	</ol>
	</li>
</ol>
2。24井板基底基质穹顶肠道 Organoids 的分离
注: 在基底矩阵圆顶内的 organoids 的形成已在别处被描述为12。
<ol>
	<li>根据制造商的指示, 将井与地下室矩阵圆顶一起涂上。</li>
	<li>用500µL 的 PBS 冲洗含基底基质圆顶的井。</li>
	<li>将冷250µL 的单元恢复解决方案 (4 ° c) 添加到每个井 (请参见材料表)。 注意: 冷细胞恢复解决方案将解所有但稳定的微管。</li>
	<li>用 P1000 微和小心地在井中上下移动, 将地下室基质从塑料中拆下, 然后将基底基体除去。</li>
	<li>在1.5 毫升低粘结离心管中收集上清液。</li>
	<li>倒置1.5 毫升低结合管几次, 并在显微镜下检查 organoids 是否已被隔离并自由移动, 而不是块状。按住显微镜, 并在低 (50X) 放大倍数下查看。</li>
	<li>用离心法将 organoids 1000 x g 进行5分钟的微丸。</li>
	<li>卸下恢复试剂并立即进行固定 (步骤 3)。</li>
</ol>
3. 孤立性肠道组织和 Organoids 的固定
<ol>
	<li>甲醇固定
	<ol>
		<li>重在-20 ° c 的1毫升甲醇中, 在10毫升和 organoids 中分离出肠窝/绒毛部分。</li>
		<li>将棺/绒毛/organoids 在-20 ° c 的冷冻室中孵育15分钟, 然后每5分钟将管子翻转。</li>
		<li>棺/绒毛/organoids 通过离心 1000 x g 进行5分钟的颗粒。</li>
		<li>取出甲醇并加入洗涤液 (1 毫升用于 organoids 和10毫升用于隔离的地穴/绒毛分数)。洗涤液可以是由 pbs 与1% 血清或 pbs 与0.1% 洗涤剂和1% 血清。 注: 使用与二次抗体相同物种的血清。例如, 如果在山羊中产生二次抗体, 则使用山羊血清。</li>
		<li>立即, 离心棺/绒毛/organoids 在 1000 x g 为5分钟的颗粒。</li>
		<li>取出洗涤液, 重棺/绒毛/organoids 在新鲜的洗涤液中。</li>
		<li>放置在一个管转子与速度设置为 20 rpm。洗涤细胞共1小时, 丸化棺/绒毛/organoids 由离心 (1000 x g 为5分钟) 每15分钟, 并更换洗涤液。 注: 如果一个管转子是不可用的, 然后每5分钟手动翻转管重隔离的文化。</li>
	</ol>
	</li>
	<li>甲醛/甲醇固定 注意: 在通风柜中处理固定和甲醛。<ol>
		<li>重在10毫升或 organoids 1 毫升的-20 ° c 定影液 (9.2 毫升的甲醇与800µL 甲醛溶液) 中分离的棺/绒毛。</li>
		<li>将棺/绒毛/organoids 在-20 ° c 的冷冻室中固定15分钟, 然后每5分钟将管子倒置。</li>
		<li>棺/绒毛/organoids 通过离心 1000 x g 进行5分钟的颗粒。</li>
		<li>去除甲醛/甲醇和添加洗涤液 (1 毫升为 organoids 或10毫升的孤立棺/绒毛分数)。洗涤液可以是由 pbs 与1% 山羊血清或 pbs 与0.1% 洗涤剂和1% 血清。</li>
		<li>离心机在 1000 x g 为5分钟的颗粒棺/绒毛/organoids。</li>
		<li>取出洗涤液和重在新鲜的洗涤液中。</li>
		<li>放置在一个管转子与速度设置为 20 rpm。洗涤细胞共1小时, 丸化棺/绒毛/organoids 由离心 (1000 x g 为5分钟) 每15分钟, 并更换洗涤液。</li>
	</ol>
	</li>
	<li>粉煤灰固定 注意: 粉煤灰粉和溶液应在通风罩中处理。 注: 大多数情况下使用 PBS 中的4% 的粉煤灰, 但根据抗体抗原性的保存, 可以使用较低的浓度。<ol>
		<li>重在10毫升4% 粉煤灰中分离出的颗粒棺/绒毛, 在 RT 中孵育 1 h, 在1毫升4% 的颗粒中分离出 organoids, 孵育30分钟。在两种情况下, 倒置管 (s) 每10分钟。</li>
		<li>棺/绒毛/organoids 通过离心 1000 x g 进行5分钟的颗粒。</li>
		<li>取出粉煤灰和添加洗涤液 (1 毫升为 organoids 和10毫升的孤立棺/绒毛)。洗涤液包括 PBS 与0.1% 洗涤剂和1% 血清。</li>
		<li>离心机在 1000 x g 为5分钟的颗粒棺/绒毛/organoids。</li>
		<li>取出洗涤液和重在新鲜的洗涤液中。</li>
		<li>放置在一个管旋转速度设置为 20 rpm。洗涤细胞共1小时, 丸化棺/绒毛/organoids 由离心 (1000 x g 为5分钟) 每15分钟, 并更换洗涤液。 注: 如果一个管转子是不可用的, 然后每5分钟手动翻转管重隔离的文化。</li>
		<li>抗原检索 (推荐的可选步骤用于粉煤灰固定)<ol>
			<li>棺/绒毛/organoids 通过离心 1000 x g 为5分钟, 除去上清液。</li>
			<li>添加 1-10 毫升的10毫米柠檬酸钠 pH 6.0 (5ml 到80° c) 和孵育在80° c 20 分钟。</li>
			<li>棺/绒毛/organoids 通过离心 1000 x g 为5分钟, 除去上清液。</li>
			<li>添加 1-10 毫升新鲜10毫米柠檬酸钠 pH 6.0 (5ml 到80° c) 和孵育在 RT 为20分钟。</li>
			<li>棺/绒毛/organoids 通过离心 1000 x g 为5分钟, 除去上清液。</li>
			<li>洗涤在 1-10 毫升 PBS 与1% 血清并且再重复3次造粒棺/绒毛/organoids 由离心 (1000 x g 为 5 min) 在加入新的洗涤的解答之前。</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
4. 阻挡步骤
<ol>
	<li>使阻塞解决方案: 在 PBS 中添加10% 次抗体种血清 (可选: 添加0.1% 洗涤剂)。</li>
	<li>棺/绒毛/organoids 通过离心 (1000 x g 为5分钟) 和去除上清液颗粒。</li>
	<li>根据所需样本数添加 5-10 毫升的阻塞解决方案 (请参阅下面的说明)。在这个阶段, 分裂棺/绒毛/organoids 溶液成单独的管 (1.5 毫升低束缚离心管与每管包含 0.2-1 毫升), 以不同的抗体染色。 注: 每个分离的棺/绒毛分数将给予 5-10 的样品, 可用于不同的标签组合取决于大小的颗粒。理想情况下, 每个样品需要 2-4 毫米之间的颗粒大小。</li></ol>在这个阶段可以组合不同的分数, 因为棺和绒毛很容易被识别 (图 2)。
	<li>在1小时的管转子的 RT 上孵育。</li>

5. 初级抗体孵化
<ol>
	<li>稀释含有10% 血清和0.1% 洗涤剂的 PBS 中的主要抗体 (见材料表)。100至200µL 主抗体溶液是每离心管样品所需的。</li>
	<li>通过离心 (1000 x g 为5分钟) 去除阻塞溶液。</li>
	<li>重的棺/绒毛/化颗粒在主抗体溶液和孵育在4° c 过夜使用管转子 (20 RPM), 以保持棺/绒毛/organoids 在悬浮。</li>
	<li>第二天: 把样品带回 RT 1 小时的管转子 (20 rpm)。</li>
	<li>用离心法 (1000 x g 为5分钟) 对样品进行颗粒分离, 去除主抗体溶液。</li>
	<li>加入1毫升的洗涤液和重的地穴/绒毛/化丸。</li>
	<li>立即通过离心 (1000 x g 为5分钟) 去除溶液。</li>
	<li>添加1毫升新鲜洗涤液和旋转的细胞在一个管转子 (20 rpm) 2 小时。通过离心每30分钟更换洗涤液, 如步骤5.7 所示。</li>
</ol>
6. 二级抗体孵化
<ol>
	<li>在 PBS 中稀释二级抗体 (见材料表), 10% 血清和0.1% 洗涤剂。每离心管样品需要200µL 的二次抗体溶液。 注: 高 cross-absorbed 抗体应用于降低小鼠窝/绒毛/化中二次抗体的反应性。</li>
	<li>离心机在 1000 x g 为5分钟的颗粒棺/绒毛/organoids 和重颗粒在200µL 的二次抗体溶液。</li>
	<li>在 RT 上旋转试管旋转管 (20 rpm) 1 小时。</li>
	<li>离心机在 1000 x g 为5分钟的颗粒棺/绒毛/organoids 和去除清 (non-bound 二次抗体)。</li>
	<li>重在洗涤液中的颗粒, 并立即离心在 1000 x g 为5分钟的颗粒棺/绒毛/organoids。</li>
	<li>在1毫升的新鲜洗涤液中除去上清和重颗粒。</li>
	<li>旋转在一个管旋转 (20 rpm) 在 RT 为 1.5-2 小时, 改变洗涤液的每 20-30 分钟, 前面所描述的步骤 6.3-6.6。</li>
</ol>
7. 核染色
<ol>
	<li>稀释洗涤液中的核污点 (按照制造商的协议), 如 DAPI 或赫斯特。例如: DAPI 库存解决方案 (20 毫克/毫升) 稀释1:10,000。该股票的解决方案可以保持在等分在-20 ° c。</li>
	<li>离心机在 1000 x g 为5分钟的颗粒棺/绒毛/organoids 和去除洗涤液。</li>
	<li>重核 counterstain 溶液中的颗粒。</li>
	<li>在 RT 上旋转一根管子 (20 rpm) 10 分钟。</li>
	<li>离心机在 1000 x g 为5分钟, 以颗粒棺/绒毛/organoids, 除去核着色液, 并重在洗涤液中的颗粒。</li>
	<li>旋转在一个管旋转 (20 rpm) 在 RT 为 30-60 分钟, 改变洗涤液每10分钟。</li>
</ol>
8. 安装隔离的棺、绒毛和 Organoids
<ol>
	<li>离心机在 1000 x g 为5分钟的颗粒棺/绒毛/organoids, 并删除所有的洗涤液。 注: 如果颗粒分散, 则再次离心。</li>
	<li>添加2滴硬设置安装介质与 antifade 剂的颗粒。</li>
	<li>削减 P200 微的结束, 并仔细重在安装媒体的颗粒。避免产生气泡。 注意: 分离的化文化非常黏稠;确保完全重。</li>
	<li>使用微在安装介质溶液中将棺/绒毛/organoids 的混合物转移到显微镜滑动中心的一条线上。 注: 该线不应长于要使用的盖玻璃。检查使用显微镜是否棺/绒毛/organoids 在幻灯片上很好地传播, 而不是成群。</li>
	<li>小心地把盖子玻璃放在上面;避免产生气泡。</li>
	<li>将玻璃幻灯片放在一本幻灯片中, 并在一夜之间存储在冰箱中, 以便在对共聚焦显微镜进行分析之前设置安装介质。 注意: 对于小鼠组织中的小鼠抗体染色, 请使用商业免疫荧光试剂盒 (参见材料表)。将阻塞步骤 (4 节) 替换为 10 min 块, 并带有蛋白质阻断液, 然后用试剂盒附带的阻挡试剂 1 h 孵化。然后在步骤 5.8 (在洗涤中间) 增加一个 1 h 孵化与荧光信号增强剂试剂。然后使用试剂盒中的荧光 dilutant (其他二次抗体也可与此试剂结合使用)。孵化为以上, 并从步骤6与其余的协议。</li>
</ol>
9. 基底基质内 Organoids 的固定和免疫标记
注: Organoids 的固定和免疫标记, 而留在地下室矩阵是产生在基底矩阵圆顶顶部的圆形玻璃片在一个24井板 (一个圆顶每井)。在24井板中, 通过对各种溶液的添加和去除, 对化基底基壳进行了处理。
<ol>
	<li>从井中取出介质并加入定影液;离开1小时</li>
	<li>在 PBS 中去除含有1% 的血清和0.1% 的洗涤剂2小时, 改变每30分钟的定影液和洗涤。</li>
	<li>用10% 的血清和0.1% 的洗涤剂去除 PBS 中的洗涤液和块, 1 小时。</li>
	<li>将 pbs 中的抗体稀释1% 血清或 pbs, 1% 血清和0.1% 洗涤剂。</li>
	<li>卸下阻塞解决方案, 并添加 100-200 μ l 主抗体 (请参见材料表) 解决方案。 注意: 重要的是要去除所有的阻断溶液, 以免进一步稀释抗体。</li>
	<li>把盘子放在冰箱里, 在4° c 的晚上孵化</li>
	<li>然后离开 RT 1-2 小时。</li>
	<li>去除抗体溶液, 洗涤 2-3 小时, 改变洗涤液每20分钟。</li>
	<li>去除所有洗涤液, 并添加 100-200 μ l 二次抗体稀释 (见材料表) 在 PBS 与1% 血清;在 RT 处孵育1小时。</li>
	<li>去除二次抗体溶液, 洗涤2小时, 每20分钟更换一次。</li>
	<li>拆卸洗涤液并添加 DAPI 溶液;在 RT 上留10分钟. 使用 DAPI 库存解决方案 (20 毫克/毫升) 稀释在1:10,000。</li></ol>该股票的解决方案可以保持在等分在-20 ° c。
	<li>移除 DAPI 溶液, 洗涤40分钟, 每10分钟更换一次。</li>
	<li>用镊子将玻璃片与基底基质圆顶一起移除, 并将其放在与基底矩阵圆顶向上的滑动面上;添加几滴安装介质, 然后将玻璃片放在顶部。</li>
	<li>将玻璃幻灯片放在一本幻灯片中, 并在一夜之间将安装介质放在冰箱中。</li>

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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+microtubule+end-binding+protein+EB2+is+a+central+regulator+of+microtubule+reorganisation+in+apico-basal+epithelial+differentiation.">The microtubule end-binding protein EB2 is a central regulator of microtubule reorganisation in apico-basal epithelial differentiation.</a> Journal of Cell Science. 126 (17), 4000-4014 (2013).</li><li>Yoneda, M., Molinolo, A. A., Ward, J. M., Kimura, S., Goodlad, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Simple+Device+to+Rapidly+Prepare+Whole+Mounts+of+the+Mouse+Intestine.">A Simple Device to Rapidly Prepare Whole Mounts of the Mouse Intestine.</a> J Vis Exp. (105), e53042 (2015).</li><li>Andersson-Rolf, A., Fink, J., Mustata, R. C., Koo, B. 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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterns+of+DNA+methylation+in+individual+colonic+crypts+reveal+aging+and+cancer-related+field+defects+in+the+morphologically+normal+mucosa.">Patterns of DNA methylation in individual colonic crypts reveal aging and cancer-related field defects in the morphologically normal mucosa.</a> Carcinogenesis. 31 (6), 1158-1163 (2010).</li><li>Whitehead, R. H., VanEeden, P. E., Noble, M. D., Ataliotis, P., Jat, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+conditionally+immortalized+epithelial+cell+lines+from+both+colon+and+small+intestine+of+adult+H-2Kb-tsA58+transgenic+mice.">Establishment of conditionally immortalized epithelial cell lines from both colon and small intestine of adult H-2Kb-tsA58 transgenic mice.</a> Proc Natl Acad Sci U S A. 90 (2), 587-591 (1993).</li><li>Hamilton, K. L., Butt, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glucose+transport+into+everted+sacs+of+the+small+intestine+of+mice.">Glucose transport into everted sacs of the small intestine of mice.</a> Adv Physiol Educ. 37 (4), 415-426 (2013).</li><li>Rogers, S. L., Rogers, G. C., Sharp, D. J., Vale, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+EB1+is+important+for+proper+assembly,+dynamics,+and+positioning+of+the+mitotic+spindle.">Drosophila EB1 is important for proper assembly, dynamics, and positioning of the mitotic spindle.</a> J Cell Biol. 158 (5), 873-884 (2002).</li></ol>

作者声明没有竞争的金融利益。

肠道组织的分离 小肠棺和绒毛和结肠棺的分离包括暴露粘膜表面, 用 EDTA 溶液处理以松弛细胞接触、分馏 (摇动) 和离心。Belshaw et al.和白石et al.对所提出的肠绒毛/隐窝隔离协议进行了修改17,18
暴露粘膜表面 我们已经尝试了一些方法, 以揭露粘膜表面的肠道在这一过程的发展。一个经典的方法是埃弗特 (转内) 管, 通常在段约100毫米长, 使用一个金属棒, 被捕获在一个折叠的组织在一端, 然后其余的管滑过管19。对于小鼠组织, 一个金属棒 (2.4 毫米直径) 与圆的末端是理想的。这种方法有利于扩大黏膜表面, 从而更好地获得 PBS 和 EDTA。我们最初使用这种方法, 但移动到切割管成短长度 (约5厘米), 并打开每个部分与解剖剪刀, 这证明更容易。这种方法是适当的, 如果只有几个肠道是必需的;但是, 如果在实验中使用更多的动物, 那么一个 purpose-built 的装置可以纵向切开管子, 如 Yoneda 的et al.所描述的那样。14将更有效。
绒毛和棺脱离肌肉层 最初, 我们使用3毫米 EDTA 在 PBS 和相对较长的孵化时间长达60分钟, 以放宽黏膜表面的基础组织17,18。在这种浓度的 EDTA, 我们发现潜伏期为30分钟, 足以松开棺从老鼠结肠。然而, 对于小肠隐窝/绒毛隔离, 我们尝试使用更多的浓缩 EDTA 较短的时间, 这证明是一种有效的方法。所有后续的工作都是用30毫米 EDTA 技术提取的组织进行的, 它们产生了绒毛或棺的相关分数。对于棺, 我们通常汇集分数 3-5 前固定, 但重要的是要检查这些是适当的分数, 因为计时将取决于一些因素, 如沿肠道的位置, 老鼠的年龄, 炎症, 以前的饮食,等同样, 在不同条件下, 在 EDTA 治疗后, 组织需要动摇的时间长短可能会有所不同。结果是含有绒毛和棺的混合物的分离组织片段, 或者主要是绒毛或棺 (图 2)。由于在结肠中没有绒毛, 因此, 在三十年代, 通过在试管中晃动组织, 可以在一步内实现隐窝提取。这些分数可以被固定和处理免疫标记。
基底基质对肠道 organoids 的分离 利用单元恢复解, 可以实现基底 organoids 的分离。该解决方案的工作原理是聚凝胶基底基体, 但温度需要 2-8 ° c。提醒注意的是, 动态微管可能不会被保存。因此, 对于动态微管的免疫标记和 + 提示, 如 EBs 细胞, 不推荐在固定前从基底基质中恢复。然而, 大多数的微管在区分化细胞是相对稳定的, 这些都被保存 (图 6)。它还有效地用于免疫标记中心和连接蛋白以及细胞标记。
固定协议 甲醛 (新鲜地由粉煤灰制成) 是一种相对快速的作用的固定剂, 形成可逆的交和4% 粉煤灰工作良好, 例如, 在免疫标记微管和伽玛蛋白和染色肌动蛋白丝与笔。更稀的粉煤灰解决方案, 如1% 工作良好的免疫标记, 例如, 与干细胞标记 Lgr5 和氏细胞标记 CD24 内的地穴干细胞利基, 而高浓度的粉煤灰没有工作。
戊二醛的加入更好地保存微管和所谓的 PHEMO 固定, 由3.7% 煤灰、0.05% 戊二醛和0.5% 洗涤剂的混合物组成在 PHEMO 缓冲 (68 毫米管子、25毫米 HEPES、15毫米 EGTA 和3毫米 MgCl2)2在不损害抗原性的情况下保护微管的优异。它也适用于免疫标记伽玛蛋白, β-蛋白, 和 e-钙黏附素, 和染色肌动蛋白丝与笔。然而, 在3D 组织和化的文化, PHEMO 固定产生不一致的结果, 因此没有使用。
甲醇是一种混凝剂固定剂, 具有较好的穿透力, 并易于保存抗原。固定与100% 甲醇 (-20 ° c) 引入了一些收缩, 给予适度的形态学保存, 和工作的微管, + 提示, 和许多中心抗体, 包括 ninein 在2D 细胞培养。然而, 在使用这种固定方法时, 有些 organoids 塌陷。此外, 通过整个棺, 绒毛, 或 organoids 的抗体渗透最初是一个问题, 但添加0.1% 洗涤剂的洗涤液和长期洗涤取得了更好的结果。
以前罗杰斯et al.使用了甲醛和甲醇的组合20到果蝇中的免疫标签 EB1 .因此, 一种基于甲醛和甲醇混合物的固定协议是基于3% 甲醛和97% 甲醇冷冻到-20 ° c 的肠道组织和 organoids, 但从罗杰斯使用的混合物中省略了5毫米碳酸钠et al。20此外, 在-20 ° c 的冷冻库中, 样品被固定。这特别适合免疫标记 + 提示, 如 CLIP-170 和 EBs, 但也证明了优良的固定和免疫标记微管和肌动蛋白在组织和 3D organoids。非常好结构保存是明显的, 并且抗原化保留了为几个骨架和伴生的蛋白质并且中心蛋白质例如伽玛蛋白并且 ninein, 虽然标号为 ninein 更加一致地使用甲醇固定.

上皮的形成与 apico-基底极性是一个基本的过程中的发展和涉及戏剧性重组的微管和中心蛋白。一个径向微管阵列从中央位于中心微管组织中心 (MTOC) 是突出的许多动物细胞, 这是非常适合于相对平坦的细胞。相比之下, 柱状上皮细胞, 如小肠, 聚集径向 transcellular 微管阵列, 更好地支持这些细胞的形状和专门功能。微管的这戏剧性的整顿达到由中心移动到尖顶和顶端 non-centrosomal MTOCs (n-MTOCs) 形成, 变得负责任为 transcellular 微管的锚地1,2,3,4,5。
我们对上皮分化和相关微管重组的认识大多来自于对不显示体内组织结构的 2D体外细胞层的调查。开发 3D在体外化文化, 由 Clevers 和同事6的先驱, 代表了一个主要的技术进步, 因为他们模仿在体内体系结构和开发。上皮分化的层次在肠道明显;在棺底部的干细胞会产生未成熟的转运增殖细胞, 随着它们迁移到小的小肠绒毛或结肠表面而逐渐分化, 它们在被流进流明7。重要的是, 这是复制在肠道 organoids, 细胞从干细胞利基增殖形成囊肿, 随后产生地穴状芽与干细胞在底部和分化逐渐走向囊肿区,它变得绒毛象8。肠道化因此代表了一个强大的模型, 不仅研究在上皮分化过程中的微管和中心重组, 但许多其他蛋白质, 以及提供一个理想的平台, 筛选药物和食品潜在的治疗优势化合物9,10。
Organoids 非常适合荧光标记蛋白质的实时成像, 基因和淘汰赛 Organoids 都可以使用 CRISPR/Cas9 基因编辑11,12生成。然而, 建立内源性蛋白质的表达和定位是非常重要的, 特别是对标记蛋白的行为进行验证。免疫标记 3D organoids 生长在基底基质或前体孤立组织比细胞生长在培养皿中的2D 更复杂。固定的协议需要保存 organoids 的微妙的3D 结构, 同时保持抗体抗原性 (即, 结合抗体的表位)。例如, 4% 甲醛 (粉煤灰) 通常用作固定剂, 但它是一个相对快速的行动固色剂, 并提供良好的形态学保存, 在我们的经验, 它经常导致丧失抗原性和不适合许多中心抗体。还应考虑固定剂和抗体穿透3D 结构和组织的能力。为此, 我们修改和开发了 3D体外组织隔离和间接免疫标记的协议 organoids 和前体内孤立的肠道组织。我们描述了如何隔离小肠棺和绒毛和结肠组织, 并包括一个协议隔离 3D organoids 作为替代固定和免疫标记在地下室矩阵。我们提出了三种替代固定协议的免疫标记的微管和中心蛋白, 如 ninein, 和微管加尾跟踪蛋白 (+ 提示), 如 EB 蛋白和 CLIP-170 (请参阅参考8, 13)。我们还讨论了与每个协议相关的优缺点。

<table>
	<tbody>
		<tr>
			<td>Dulbecco&#8217;s Phosphate Buffered Saline (PBS)</td>
			<td>Sigma</td>
			<td>D8537-500ML</td>
			<td>washing and PFA</td>
		</tr>
		<tr>
			<td>Cell Recovery Solution</td>
			<td>Corning</td>
			<td>354253</td>
			<td>isolate organoids</td>
		</tr>
		<tr>
			<td>lobind microcentrifuge tubes</td>
			<td>Eppendorf</td>
			<td>30108116</td>
			<td>prevent cells sticking</td>
		</tr>
		<tr>
			<td>0.5 M EDTA solution, pH 8.0</td>
			<td>Sigma</td>
			<td>03690-100ML</td>
			<td>Crypt isolation</td>
		</tr>
		<tr>
			<td>70 &#956;m cell strainer</td>
			<td>Fisher Scientific</td>
			<td>11517532</td>
			<td>Isolate crypts from villi</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma</td>
			<td>T8787</td>
			<td>detergent</td>
		</tr>
		<tr>
			<td>goat serum</td>
			<td>Sigma</td>
			<td>G6767</td>
			<td>blocking agent</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Sigma</td>
			<td></td>
			<td>used as non-stick agent</td>
		</tr>
		<tr>
			<td>Paraformaldehyde, 4% in PBS</td>
			<td>Alfa Aesar</td>
			<td>J61899</td>
			<td>fixative</td>
		</tr>
		<tr>
			<td>Formaldehyde solution (36.5&#8211;38% in H2O)</td>
			<td>Sigma</td>
			<td>F8775</td>
			<td>used as fixative</td>
		</tr>
		<tr>
			<td>Methanol 99.9% Analytical grade</td>
			<td>Fisher</td>
			<td>M/4000/17</td>
			<td>used as fixative</td>
		</tr>
		<tr>
			<td>MaxFluor Mouse on Mouse Immunofluorescence Detection Kit</td>
			<td>Maxvision 
			Biosciences</td>
			<td>MF01-S</td>
			<td>commercial immunofluorescence kit; to reduce non-specific labelling</td>
		</tr>
		<tr>
			<td>Rotator SB2 or SB3</td>
			<td>Stuart</td>
			<td></td>
			<td>microcentrifuge tube rotator</td>
		</tr>
		<tr>
			<td>Sodium citrate</td>
			<td></td>
			<td></td>
			<td>antigen retrieval</td>
		</tr>
		<tr>
			<td>Hydromount</td>
			<td>National Diagnostics</td>
			<td>HS-106</td>
			<td>mountant</td>
		</tr>
		<tr>
			<td>DABCO - 1,4-diazabicyclo[2.2.2]octane</td>
			<td>Sigma</td>
			<td>D27802</td>
			<td>anti-fade agent</td>
		</tr>
		<tr>
			<td>Permanent Positive Charged, Pre-Washed, 90 Degree Ground Edges</td>
			<td>Clarity</td>
			<td>N/C366</td>
			<td>slides</td>
		</tr>
		<tr>
			<td>Glass coverslip - thickness no. 1, 22 x 50 mm</td>
			<td>Clarity</td>
			<td>NQS13/2250</td>
			<td>coverslips</td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>Corning</td>
			<td>356231</td>
			<td>Basement matrix for 3D organoid culture</td>
		</tr>
		<tr>
			<td>50 mL conical tubes</td>
			<td>Sarstedt</td>
			<td>62.547.254</td>
			<td>for processing tissue/organoids</td>
		</tr>
		<tr>
			<td>15 mL conical tubes</td>
			<td>Sarstedt</td>
			<td>62.554.502</td>
			<td>for processing tissue/organoids</td>
		</tr>
		<tr>
			<td>1.5 mL LoBind tubes</td>
			<td>Eppendorf</td>
			<td>30108051</td>
			<td>for isolated organoids</td>
		</tr>
		<tr>
			<td>Mouse monoclonal anti-E-cadherin antibody</td>
			<td>BD Biosciences</td>
			<td>610181</td>
			<td>primary antibody 1:500</td>
		</tr>
		<tr>
			<td>Rat monoclonal anti-tubulin YL1/2 antibody</td>
			<td>Abcam</td>
			<td>ab6160</td>
			<td>primary antibody 1:100</td>
		</tr>
		<tr>
			<td>Rabbit alpha-tubulin antibody</td>
			<td>Abcam</td>
			<td>ab15246</td>
			<td>primary antibody 1:100</td>
		</tr>
		<tr>
			<td>Rabbit anti-beta-actin antibody</td>
			<td>Abcam</td>
			<td>ab8227</td>
			<td>primary antibody 1:100</td>
		</tr>
		<tr>
			<td>Rat monoclonal anti-p150Glued antibody</td>
			<td>BD Bioscience</td>
			<td>610473/4</td>
			<td>primary antibody 1:100</td>
		</tr>
		<tr>
			<td>Rat monoclonal EB3KT36 antibody</td>
			<td>Abcam</td>
			<td>ab53360</td>
			<td>primary antibody 1:200</td>
		</tr>
		<tr>
			<td>Mouse monoclonal EB1 antibody</td>
			<td>BD Bioscience</td>
			<td>610535</td>
			<td>primary antibody 1:200</td>
		</tr>
		<tr>
			<td>Rabbit anti-Lgr5 antibody</td>
			<td>Abgent</td>
			<td>AP2745d</td>
			<td>primary antibody 1:100</td>
		</tr>
		<tr>
			<td>Dylight anti-rat 488</td>
			<td>Jackson</td>
			<td>112545167</td>
			<td>seconday antibody 1:400</td>
		</tr>
		<tr>
			<td>Dylight anti-mouse 488</td>
			<td>Jackson</td>
			<td>ST115545166</td>
			<td>seconday antibody 1:400</td>
		</tr>
		<tr>
			<td>Dylight anti-rabbit 647</td>
			<td>Jackson</td>
			<td>111605144</td>
			<td>seconday antibody 1:400</td>
		</tr>
	</tbody>
</table>

immuno-fluorescent labeling of microtubules and centrosomal proteins in ex vivo intestinal tissue and 3d in vitro intestinal organoids

3D体外organoids 的出现, 模仿体内组织结构和形态发生, 极大地提高了研究细胞和发育生物学关键生物学问题的能力。此外, organoids 与最近基因编辑和病毒基因递送技术的进步一起, 有望推动医学研究和开发新的治疗疾病的药物。Organoids 生长的体外基底基质为研究各种蛋白质的行为和功能提供了强大的模型系统, 非常适合荧光标记蛋白的活体成像。但是, 在 体外组织中建立内源性蛋白的表达和定位, 并在 organoids 中, 对标记蛋白的行为进行验证是很重要的。为此, 我们已经开发和修改了组织隔离, 固定和免疫标记协议的微管, 中心, 和相关的蛋白在前体肠道组织和在外肠道 organoids。其目的是保存 organoids/组织的3D 体系结构, 同时保持抗体的抗原性, 使固定剂和抗体有良好的穿透和清除能力。接触冷聚所有但稳定的微管, 这是一个关键因素时, 修改各种协议。我们发现, 增加乙酸酸 (edta) 的浓度从3毫米到30毫米提供了有效的剥离绒毛和棺在小肠, 而3毫米 edta 是足够的结肠棺。开发的甲醛/甲醇固定协议给予了很好的结构保存, 同时也保持了抗原性的有效标记微管, 肌动蛋白, 和末端结合 (EB) 蛋白。它也工作的中心蛋白 ninein, 虽然甲醇协议的工作更一致。我们进一步证实, 微管和相关蛋白的固定和免疫标记可以通过基底基质中的 organoids 分离或保持。

免疫标记肠道组织的分离 所描述的结肠和小肠组织隔离协议是针对微管和相关蛋白的保存和免疫标记而优化的, 但不适用于干细胞生存能力和化生成 (图 1和表 1).其目的是产生尽可能清洁 (无粘液和其他组织) 的地穴和绒毛派系, 同时尽量减少对 EDTA 和寒冷的接触, 以保持结构, 防止降解的微管与冰冷溶液, 从而导致降解的所有, 但稳定的微管。图 2显示了来自孤立小肠组织的分数2和3的图像的示例, 分数2包含了绒毛和棺的混合 (图 2A、B), 而分数3主要包含棺 (图 2C 、D)。
孤立性肠组织的固定与免疫标记 个体或组合的分数, 然后处理固定和免疫标记通过一系列步骤, 包括固定, 洗涤剂, 阻断, 抗体和洗涤解决方案, 之前 re-suspending 最终绒毛/棺颗粒在安装媒体,转移到幻灯片, 并覆盖玻璃片。棺和绒毛在共聚焦显微镜下成像。
良好的保存和标签的微管和肌动蛋白在绒毛和棺是通过以下方式实现: 甲醛/甲醇固定在-20 ° c, 在 pbs 中重复洗涤含有0.1% 洗涤剂和1% 的血清和0.1% 的 pbs 阻断洗涤剂和10% 血清, 其次是夜间孵化在4° c 的主要抗体, 然后 2 h 在二级抗体室温 (图 3)。甲醛/甲醇固定也很好的标记 + 提示, 如 EBs 和 CLIP-170 在孤立的棺和绒毛 (图 4)。在棺 (图 4A) 中, 微管的 EB3 堆积 (称为彗星) 是明显的, 而沿稳定微管晶格的关联可以在绒毛样本中看到 (图 4C)。CLIP-170 和 p150胶合(亚单位的 dynactin) 明显地明显位于孤立绒毛的顶端 n-MTOCs (图 4B)。固定与甲醛/甲醇协议没有一贯工作的 ninein 定位在孤立的肠道组织使用我们的 Pep3 抗体对小鼠 ninein。然而, 甲醇固定在-20 ° c 之后与甲醛/甲醇相同的洗涤和堵塞解决方案, 使 ninein 在孤立的棺和绒毛 (图 5; 参考8) 中的定位非常好。有趣的是, 当 ninein 集中在顶端体的时候, 细胞基础的一些积累在隔离棺 (图 5) 中的一些细胞中是明显的。这是否由于非特异性标记或隔离程序延迟固定 (从而影响保存) 的结果, 将需要进一步调查。然而, 甲醇固定的 (-20 ° c) 低温部分绒毛 (参见图 3bi在8) 也发现了 ninein 在细胞基地在一些细胞暗示 ninein 可能也联想与一个基本的微管的人口。
基底基质分离 organoids 的固定与免疫标记 小肠 organoids 在基底基质中生成并生长三周或更长 (图 6A; 参考6,15)。采用冷 (4 ° c) 细胞回收液将 organoids 从基底基质中分离出来。聚基底基质溶液 organoids 在固定和免疫标记前转移到管和离心。这产生了非常干净的准备, 并允许良好的访问 organoids 的各种解决方案。化绒毛域内的分化细胞含有稳定的 apico 基微管;这些标记在大多数情况下 (图 6B, C) 和 EB1 也可以看到沿微管晶格 (图 6E; 参考13)。然而, 冷细胞恢复解决方案可能导致降解的动态微管, 这是明显的, 在一些样本的缺乏 EB1 彗星 (绑定到增加端的微管) 在基底地穴域 (图6F).在其他样品中, 星体 (动态) 微管被保存 (图 6D)。化隔离之前的固定和免疫标记也工作的连接蛋白, ninein, CLIP-170, 和细胞标记, 如粘蛋白为杯状细胞和嗜 A enteroendocrine 细胞。
基底基质内 organoids 的固定与免疫标记 图 7显示一个化在囊肿阶段 (一个C) 和在早期的地穴开发 (D), 两者都固定在甲醛/甲醇和免疫标记的微管和 ninein。良好的微管保存和标记, 以及在顶端 n-MTOCs ninein 标记是明显的。图 8A, B 显示了一天内的地穴域6化固定与甲醇协议, 并标记为微管和 EB1。良好的保存微管和 EB1 彗星是明显的建议保存的动态微管。
Organoids 也固定和免疫标记, 而留在地下室矩阵。这一过程的缺点可能是在基底基质 (图 8B) 中的固定剂和捕集抗体的渗透率很低, 但在两种情况下, 当0.1% 洗涤剂被包括在定影液和/或洗涤溶液中时, 不太频繁。此外, 4% 的粉煤灰并没有保存地下室的良好, 但导致它解散, 虽然这是不那么与1% 粉煤灰。甲醇固定, 另一方面, 有时诱发化崩溃。
标签与一些抗体, 如对干细胞标记 Lgr5 和氏细胞标记 CD24 证明不成功的4% 粉煤灰, 甲醇, 或甲醛/甲醇协议。然而, 用0.1% 的洗涤剂在室温下用1% 的 organoids 在 PBS 中固定基底基质中的 Lgr5, 这就导致了对 CD24 (图 9) 的标记。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56662/56662fig1.jpg"> 
图 1: 小肠绒毛和棺的分离.在固定和免疫标记之前, 小肠绒毛和隐窝隔离的关键步骤的流程图。<a href="//ecsource.jove.com/files/ftp_upload/56662/56662fig1large.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/56662/56662fig2.jpg"> 
图 2: 从小鼠小肠分离的绒毛和棺.明显微镜图像的肠道分数显示绒毛 (大箭头) 和棺 (小箭头)。(A, B)分数2包含了绒毛和棺的混合物, 并保存在绒毛和地穴上的形态学在B中很明显。(C, D)分数3显示了分离的棺和缺乏绒毛和完整的棺包括一个分岔地穴在C。刻度条 = 500 μ m (A, C);100μ m (B, D)。<a href="//ecsource.jove.com/files/ftp_upload/56662/56662fig2large.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/56662/56662fig3.jpg"> 
图 3: 孤立的小肠绒毛和固定在甲醛/甲醇和免疫标记的微管和肌动蛋白的地穴.(A) 绒毛和地穴上皮的示意图以不同的细胞类型表明。突出显示的框指示在B和C中映像的代表区域。(B, C)共焦光学剖面通过部分绒毛 (B) 和基底地穴 (C) 从小肠分离, 使用30毫米 EDTA 并固定在甲醛/甲醇中, 在 pbs 中含有1% 山羊血清和0.1% 洗涤剂, 在 pbs 中被堵塞含有10% 山羊血清和0.1% 洗涤剂, 并标记为微管与大鼠单克隆 anti-tubulin 抗体 (绿色) 和肌动蛋白与兔多克隆抗β肌动蛋白抗体 (红)。保存完好的 apico 基微管束在绒毛和隐窝细胞中都很明显, 肌动蛋白可以被发现集中在朝向管腔 (箭头) 的顶端区域。缩放条形图 = 5 μ m。<a href="//ecsource.jove.com/files/ftp_upload/56662/56662fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56662/56662fig4.jpg"> 
图 4: 在甲醛/甲醇中固定的孤立小肠窝和绒毛, 免疫标记用于微管、EB3、p150粘附和 CLIP-170.用30毫米 EDTA 和固定在甲醛/甲醇中, 在 pbs 中洗净含有10% 山羊血清和0.1% 洗涤剂的地下墓穴和绒毛区的共焦光学部分, 在 pbs 中含有10% 山羊血清和0.1% 洗涤剂, 并免疫标记。(A) 用兔多克隆α-蛋白抗体 (红色) 和大鼠单克隆 EB3KT36 抗体 (绿色) 标记的地穴, DAPI (蓝色) 显示 apico 基微管和 EB3 彗星的 DNA 染色。倒置的单声道图像清楚地显示 EB3 的彗星贯穿于基底隐窝细胞中, 这意味着良好的动态保存以及稳定的微管。(B) 用兔多克隆 CLIP-170 抗体标记的绒毛上皮细胞 (红色, 参见参考16) 和小鼠单克隆 p150 抗体 (绿色) 显示顶端共存.倒置的单声道图像如下图所示。(C) 用兔多克隆α-蛋白抗体 (红色) 和大鼠单克隆 EB3-KT36 抗体 (绿色) 标记的绒毛细胞, 用 DAPI (蓝色) 显示 apico-基底微管与 EB3 沿晶格的 DNA 染色。在放大后的图像中突出显示了 EB3 晶格关联, 而反转的单通道图像则同时显示 EB3 彗星和晶格关联。缩放条形图 = 5 μ m。<a href="//ecsource.jove.com/files/ftp_upload/56662/56662fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56662/56662fig5.jpg"> 
图 5: 固定在甲醇中的孤立结肠隐窝, 免疫标记为 ninein 和 e-钙黏蛋白, 染色与 DAPI.从结肠分离的基底和中转放大区的共焦光学部分使用3毫米 EDTA 和固定在甲醇中, 在 pbs 中洗涤, 含有1% 山羊血清和0.1% 洗涤剂, 在 pbs 中含有10% 山羊血清和0.1% 洗涤剂。地窖被标记了与兔子多克隆 ninein 抗体 (Pep3, 也参见参考8, 红色) 和小鼠单克隆 e-钙黏蛋白抗体 (绿色) 和沾染与 DAPI (蓝色)。反转单通道图像仅显示 ninein。图像显示了一个保存完好的地穴与 e-钙黏附素揭示的个别细胞和 ninein 的轮廓集中在顶端体。提示有良好的固定剂和抗体的穿透力和抗原性的保存。刻度条 = 5 μ m。<a href="//ecsource.jove.com/files/ftp_upload/56662/56662fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/56662/56662fig6.jpg"> 
图 6: 化的开发、固定和免疫标记孤立的 organoids.(A) 相衬图像显示不同阶段的化发育从细胞聚集到囊肿与芽启动和完全形成 organoids 与隐窝和绒毛领域。(B-F)利用4° c (10 分钟) 的细胞恢复溶液, 在含有10% 山羊血清和0.1% 洗涤剂的 pbs 中洗涤, 在含10% 只山羊的 pbs 中进行阻断, 通过 organoids 从基底基质分离出共焦光学剖面血清和0.1% 洗涤剂, 和免疫标签的微管, β-蛋白, 和 EB1。(B) 化囊肿标记为微管 (蓝色) 和β-蛋白 (红色), 在大多数细胞中显露良好的微管保存和标记。(C) 不同的 apico 基底微管是明显的, 这些扩大上皮细胞从化囊肿。(D) 将标记为微管的单元格划分为包括星光 (动态) 微管 (箭头) 的心轴。(E, F)绒毛域 (E) 和隐窝域 (F) 化区域显示一些 EB1 标记沿着稳定的微管的晶格, 特别是在绒毛, 而很少 EB1 彗星被看见甚而在基础地穴之内暗示那动态微管尚未保存。缩放条形图 = 20 μ m (A);2μ m (D);5μ m (B, C, E-F)。<a href="//ecsource.jove.com/files/ftp_upload/56662/56662fig6large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/56662/56662fig7.jpg"> 
图 7: Organoids 固定在甲醛/甲醇和免疫标记的基底基质中的微管和 ninein.organoids 固定在甲醛/甲醇中的共焦光学剖面, 在 PBS 中含有10% 山羊血清和0.1% 洗涤剂, 并在基底基质中进行标记。(A-C)化囊肿标记为微管 (绿色) 和 ninein (Pep3; 参考8, 红色), 并沾有 DAPI (蓝色) 显示合并图像在A和反转单通道图像的微管 (B) 和 ninein (C)。图像显示 apico 基微管和顶端 ninein 定位, 建议非常好的结构保存的化和渗透抗体以及清除未绑定的抗体。(D, E)化与开发的地穴固定和标记为上述和再次显示优秀的结构保存, 标记和清除抗体。明显的 apico-基底微管和顶端 n-MTOC ninein 定位在D中的盒装区域的放大图像 (E) 中是显而易见的, 并突出显示。刻度条 = 10 微米 (A-D);5μ m (E)。<a href="//ecsource.jove.com/files/ftp_upload/56662/56662fig7large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/56662/56662fig8.jpg"> 
图 8: Organoids 固定在甲醇和免疫标记的基底基质中的微管和 EB1.organoids 固定在甲醇中的共焦光学剖面, 在 PBS 中被洗涤和堵塞, 含有10% 山羊血清和0.1% 洗涤剂, 并标记, 同时留在基底基质中。(a) 囊肿域从一个完全发达的化标记与兔多克隆α蛋白 (红色) 和小鼠单克隆 EB1 (绿色) 抗体显示 apico 基微管, 纺锤 (箭头) 在两个分裂细胞和不同的 EB1 彗星。一些捕获的未绑定的 EB1 抗体是明显的。但是, 对微管和 EB1 的结构保存和标记也有良好的观察。EB1 彗星的存在表明, 动态微管已被保存 (A, 反转)。(B) 化囊肿区的反转图像, 显示α-蛋白抗体标记, 并在周围基底基质 (箭头) 内有相当多的抗体。缩放条形图 = 5 μ m。<a href="//ecsource.jove.com/files/ftp_upload/56662/56662fig8large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/56662/56662fig9.jpg"> 
图 9: Organoids 固定在基底基质内的1% 粉煤灰和免疫标记的 Lgr5 和 CD24.organoids 的共焦光学剖面在1% 的 pbs 中固定在基底基质中, 其中含有0.1% 洗涤剂, 在 pbs 中用1% 山羊血清和0.1% 洗涤剂洗涤, 并用抗体对 Lgr5 和 CD24 进行标记。(A, B)在一个地穴域内的干细胞利基显示氏细胞阳性的 CD24 (红色)。共焦和相位对比度图像已在A中合并。B显示 CD24 标记的单个通道。(C) 在隐窝域内显示 Lrg5 阳性干细胞的干细胞区。缩放条形图 = 10 μ m。<a href="//ecsource.jove.com/files/ftp_upload/56662/56662fig9large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Table 1" src="/files/ftp_upload/56662/56662table1.jpg"> 
表 1: 小肠隐窝和绒毛隔离和固定的时间轴.<a href="//ecsource.jove.com/files/ftp_upload/56662/56662table1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Immuno-fluorescent Labeling of Microtubules and Centrosomal Proteins in Ex Vivo Intestinal Tissue and 3D In Vitro Intestinal Organoids at JoVE.com

Immuno-fluorescent Labeling of Microtubules and Centrosomal Proteins in Ex Vivo Intestinal Tissue and 3D In Vitro Intestinal Organoids

我们提出了从体内组织和体外基底基质 organoids 分离肠道3D 结构的协议, 并详细介绍了针对微管免疫标记优化的不同固定和染色方案,中心, 和连接蛋白以及细胞标记包括干细胞蛋白 Lgr5。

免疫荧光标记的微管和中心蛋白在体内肠道组织和 3D体外肠道 Organoids

The advent of 3D in vitro organoids that mimic the in vivo tissue architecture and morphogenesis has greatly advanced the ability to study key biological ...

immuno-fluorescent-labeling-microtubules-centrosomal-proteins-ex-vivo

Research

JoVE Journal

Developmental Biology

Immuno-fluorescent Labeling of Microtubules and Centrosomal Proteins in Ex Vivo Intestinal Tissue and 3D In Vitro Intestinal Organoids

15.2K 次观看.  University of East Anglia. 我们提出了从体内组织和体外基底基质 organoids 分离肠道3D 结构的协议, 并详细介绍了针对微管免疫标记优化的不同固定和染色方案,中心, 和连接蛋白以及细胞标记包括干细胞蛋白 Lgr5。

视频: Immuno-fluorescent Labeling of Microtubules and Centrosomal Proteins in Ex Vivo Intestinal Tissue and 3D In Vitro Intestinal Organoids

Viral-mediated Labeling and Transplantation of Medial Ganglionic Eminence (MGE) Cells for In Vivo Studies

GABAergic cortical interneurons, derived from the embryonic medial and caudal ganglionic eminences (MGE and CGE), are functionally and morphologically diverse. Inroads have been made in understanding the roles of distinct cortical interneuron subgroups, however, there are still many mechanisms to be worked out that may contribute to the development and maturation of different types of GABAergic cells. Moreover, altered GABAergic signaling may contribute to phenotypes of autism, schizophrenia and epilepsy. Specific Cre-driver lines have begun to parcel out the functions of unique interneuron subgroups. Despite the advances in mouse models, it is often difficult to efficiently study GABAergic cortical interneuron progenitors with molecular approaches in vivo. One important technique used to study the cell autonomous programming of these cells is transplantation of MGE cells into host cortices. These transplanted cells migrate extensively, differentiate, and functionally integrate. In addition, MGE cells can be efficiently transduced with lentivirus immediately prior to transplantation, allowing for a multitude of molecular approaches. Here we detail a protocol to efficiently transduce MGE cells before transplantation for in vivo analysis, using available Cre-driver lines and Cre-dependent expression vectors. This approach is advantageous because it combines precise genetic manipulation with the ability of these cells to disperse after transplantation, permitting greater cell-type specific resolution in vivo.

Viral-mediated Labeling and Transplantation of Medial Ganglionic Eminence MGE Cells for In Vivo Studies

GABAergic cortical interneuron progenitors disperse, develop and synaptically integrate into a host cortex after transplantation. These cells can be easily transduced before transplantation for in vivo studies of genetically modified GABAergic precursors. Here, we show viral labeling techniques to target specific interneuron subgroups using existing Cre lines and Cre-dependent reporters.

病毒介导的标签和内侧神经节隆起移植（MGE）细胞<em&gt;在体内</em&gt;研究

GABAergic cortical interneurons, derived from the embryonic medial and caudal ganglionic eminences (MGE and CGE), are functionally and morphologically ...

科研

发育生物学

Characterization of Thymic Settling Progenitors in the Mouse Embryo Using In Vivo and In Vitro Assays

Characterizing thymic settling progenitors is important to understand the pre-thymic stages of T cell development, essential to devise strategies for T cell replacement in lymphopenic patients. We studied thymic settling progenitors from murine embryonic day 13 and 18 thymi by two complementary in vitro and in vivo techniques, both based on the &#8220;hanging drop&#8221; method. This method allowed colonizing irradiated fetal thymic lobes with E13 and/or E18 thymic progenitors distinguished by CD45 allotypic markers and thus following their progeny. Colonization with mixed populations allows analyzing cell autonomous differences in biologic properties of the progenitors while colonization with either population removes possible competitive selective pressures. The colonized thymic lobes can also be grafted in immunodeficient male recipient mice allowing the analysis of the mature T cell progeny in vivo, such as population dynamics of the peripheral immune system and colonization of different tissues and organs. Fetal thymic organ cultures revealed that E13 progenitors developed rapidly into all mature CD3+ cells and gave rise to the canonical &#947;&#948; T cell subset, known as dendritic epithelial T cells. In comparison, E18 progenitors have a delayed differentiation and were unable to generate dendritic epithelial T cells. The monitoring of peripheral blood of thymus-grafted CD3-/- mice further showed that E18 thymic settling progenitors generate, with time, larger numbers of mature T cells than their E13 counterparts, a feature that could not be appreciated in the short term fetal thymic organ cultures.

Characterization of Thymic Settling Progenitors in the Mouse Embryo Using In Vivo and In Vitro Assays

This article describes in vivo and in vitro methodology to characterize the thymic settling progenitors by the analysis of the kinetics of generation, phenotype and numbers of their T cell progeny.

胸腺表征祖定居在小鼠胚胎使用<em&gt;在体内</em&gt;和<em&gt;在体外</em&gt;测定

Characterizing thymic settling progenitors is important to understand the pre-thymic stages of T cell development, essential to devise strategies for T ...

In Vitro and In Vivo Models to Study Corneal Endothelial-mesenchymal Transition

Corneal endothelial cells (CECs) play a crucial role in maintaining corneal clarity through active pumping. A reduced CEC count may lead to corneal edema and diminished visual acuity. However, human CECs are prone to compromised proliferative potential. Furthermore, stimulation of cell growth is often complicated by gradual endothelial-mesenchymal transition (EnMT). Therefore, understanding the mechanism of EnMT is necessary for facilitating the regeneration of CECs with competent function. In this study, we prepared a primary culture of bovine CECs by peeling the CECs with Descemet's membrane from the corneal button and demonstrated that bovine CECs exhibited the EnMT process, including phenotypic change, nuclear translocation of &#946;-catenin, and EMT regulators snail and slug, in the in vitro culture. Furthermore, we used a rat corneal endothelium cryoinjury model to demonstrate the EnMT process in vivo. Collectively, the in vitro primary culture of bovine CECs and in vivo rat corneal endothelium cryoinjury models offers useful platforms for investigating the mechanism of EnMT.

In Vitro and In Vivo Models to Study Corneal Endothelial-mesenchymal Transition

A primary culture of bovine corneal endothelial cells was used to investigate the mechanism of corneal endothelial-mesenchymal transition. Furthermore, a rat corneal endothelium cryoinjury model was used to demonstrate corneal endothelial-mesenchymal transition in vivo.

在体外和体内模型来研究角膜内皮-间充质转换

Corneal endothelial cells (CECs) play a crucial role in maintaining corneal clarity through active pumping. A reduced CEC count may lead to corneal edema ...

Live Imaging of Mouse Secondary Palate Fusion

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to cleft secondary palate, a common congenital anomaly in humans. Secondary palate fusion has been extensively studied leading to several proposed cellular mechanisms that may mediate this process. However, these studies have been mostly performed on fixed embryonic tissues at progressive timepoints during development or in fixed explant cultures analyzed at static timepoints. Static analysis is limited for the analysis of dynamic morphogenetic processes such a palate fusion and what types of dynamic cellular behaviors mediate palatal fusion is incompletely understood. Here we describe a protocol for live imaging of ex vivo secondary palate fusion in mouse embryos. To examine cellular behaviors of palate fusion, epithelial-specific Keratin14-cre was used to label palate epithelial cells in ROSA26-mTmGflox reporter embryos. To visualize filamentous actin, Lifeact-mRFPruby reporter mice were used. Live imaging of secondary palate fusion was performed by dissecting recently-adhered secondary palatal shelves of embryonic day (E) 14.5 stage embryos and culturing in agarose-containing media on a glass bottom dish to enable imaging with an inverted confocal microscope. Using this method, we have detected a variety of novel cellular behaviors during secondary palate fusion. An appreciation of how distinct cell behaviors are coordinated in space and time greatly contributes to our understanding of this dynamic morphogenetic process. This protocol can be applied to mutant mouse lines, or cultures treated with pharmacological inhibitors to further advance understanding of how secondary palate fusion is controlled.

Here, we present a protocol for live imaging of mouse secondary palate fusion using confocal microscopy. This protocol can be used in combination with a variety of fluorescent reporter mouse lines, and with pathway inhibitors for mechanistic insight. This protocol can be adapted for live imaging in other developmental systems.

小鼠第二腭融合的实时成像

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to ...

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

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the proteins involved in these pathways are evolutionarily conserved between mammals and other eukaryotes, such as the fruit fly Drosophila melanogaster, suggesting that similar organizing principles exist during the development of these organisms. Importantly, Drosophila has been used extensively to identify cellular and molecular mechanisms regulating processes that are required in mammals including neurogenesis, differentiation, axonal guidance, and synaptogenesis. Flies have also been used successfully to model a variety of human neurodevelopmental diseases. Here we describe a protocol for the step-by-step microdissection, fixation, and immunofluorescent localization of proteins within the adult Drosophila brain. This protocol focuses on two example neuronal populations, mushroom body neurons and retinal photoreceptors, and includes optional steps to trace individual mushroom body neurons using Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. Example data from both wild-type and mutant brains are shown along with a brief description of a scoring criteria for axonal guidance defects. While this protocol highlights two well-established antibodies for investigating the morphology of mushroom body and photoreceptor neurons, other Drosophila brain regions and the localization of proteins within other brain regions can also be investigated using this protocol.

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

This protocol describes the dissection and immunostaining of adult Drosophila melanogaster brain tissues. Specifically, this protocol highlights the use of Drosophila mushroom body and photoreceptor neurons as example neuronal subsets that can be accurately used to uncover general principles underlying many aspects of neuronal development.

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 ...

Processing of Human Cardiac Tissue Toward Extracellular Matrix Self-assembling Hydrogel for In Vitro and In Vivo Applications

Acellular extracellular matrix preparations are useful for studying cell-matrix interactions and facilitate regenerative cell therapy applications. Several commercial extracellular matrix products are available as hydrogels or membranes, but these do not possess tissue-specific biological activity. Because perfusion decellularization is usually not possible with human heart tissue, we developed a 3-step immersion decellularization process. Human myocardial slices procured during surgery are first treated with detergent-free hyperosmolar lysis buffer, followed by incubation with the ionic detergent, sodium dodecyl sulfate, and the process is completed by exploiting the intrinsic DNase activity of fetal bovine serum. This technique results in cell-free sheets of cardiac extracellular matrix with largely preserved fibrous tissue architecture and biopolymer composition, which were shown to provide specific environmental cues to cardiac cell populations and pluripotent stem cells. Cardiac extracellular matrix sheets can then be further processed into a microparticle powder without further chemical modification, or, via short-term pepsin digestion, into a self-assembling cardiac extracellular matrix hydrogel with preserved bioactivity.

Processing of Human Cardiac Tissue Toward Extracellular Matrix Self-assembling Hydrogel for In Vitro and In Vivo Applications

This protocol describes the complete decellularization of human myocardium while preserving its extracellular matrix components. Further processing of the extracellular matrix results in the production of microparticles and a cytoprotective self-assembling hydrogel.

人心脏组织对细胞外基质自组装水凝胶的处理体外和体内应用

Acellular extracellular matrix preparations are useful for studying cell-matrix interactions and facilitate regenerative cell therapy applications. ...

Real-time Measurement of Epithelial Barrier Permeability in Human Intestinal Organoids

Advances in 3D culture of intestinal tissues obtained through biopsy or generated from pluripotent stem cells via directed differentiation, have resulted in sophisticated in vitro models of the intestinal mucosa. Leveraging these emerging model systems will require adaptation of tools and techniques developed for 2D culture systems and animals. Here, we describe a technique for measuring epithelial barrier permeability in human intestinal organoids in real-time. This is accomplished by microinjection of fluorescently-labeled dextran and imaging on an inverted microscope fitted with epifluorescent filters. Real-time measurement of the barrier permeability in intestinal organoids facilitates the generation of high-resolution temporal data in human intestinal epithelial tissue, although this technique can also be applied to fixed timepoint imaging approaches. This protocol is readily adaptable for the measurement of epithelial barrier permeability following exposure to pharmacologic agents, bacterial products or toxins, or live microorganisms. With minor modifications, this protocol can also serve as a general primer on microinjection of intestinal organoids and users may choose to supplement this protocol with additional or alternative downstream applications following microinjection.

This protocol describes the measurement of epithelial barrier permeability in real-time following pharmacologic treatment in human intestinal organoids using fluorescent microscopy and live cell microscopy.

人肠道 Organoids 上皮屏障通透性的实时测量

Advances in 3D culture of intestinal tissues obtained through biopsy or generated from pluripotent stem cells via directed differentiation, have resulted ...

Genetic Engineering of Primary Mouse Intestinal Organoids Using Magnetic Nanoparticle Transduction Viral Vectors for Frozen Sectioning

Intestinal organoid cultures provide a unique opportunity to investigate intestinal stem cell and crypt biology in vitro, although efficient approaches to manipulate gene expression in organoids have made limited progress in this arena. While CRISPR/Cas9 technology allows for precise genome editing of cells for organoid generation, this strategy requires extensive selection and screening by sequence analysis, which is both time-consuming and costly. Here, we provide a detailed protocol for efficient viral transduction of intestinal organoids. This approach is rapid and highly efficient, thus decreasing the time and expense inherent in CRISPR/Cas9 technology. We also present a protocol to generate frozen sections from intact organoid cultures for further analysis with immunohistochemical or immunofluorescent staining, which can be used to confirm gene expression or silencing. After successful transduction of viral vectors for gene expression or silencing is achieved, intestinal stem cell and crypt function can be rapidly assessed. Although most organoid studies employ in vitro assays, organoids can also be delivered to mice for in vivo functional analyses. Moreover, our approaches are advantageous for predicting therapeutic responses to drugs because currently available therapies generally function by modulating gene expression or protein function rather than altering the genome.

We describe step-by-step instructions to: 1) efficiently engineer intestinal organoids using magnetic nanoparticles for lenti- or retroviral transduction, and, 2) generate frozen sections from engineered organoids. This approach provides a powerful tool to efficiently alter gene expression in organoids for investigation of downstream effects.

使用磁纳米粒子转导病毒载体进行冷冻切片的原小鼠肠道器官的遗传工程

Intestinal organoid cultures provide a unique opportunity to investigate intestinal stem cell and crypt biology in vitro, although efficient approaches to ...

Visualizing the Node and Notochordal Plate In Gastrulating Mouse Embryos Using Scanning Electron Microscopy and Whole Mount Immunofluorescence

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is the formation of the transient organizers, the node and notochordal plate, from cells that have passed through the primitive streak. The proper formation of these signaling centers is essential for the development of the body plan and techniques to visualize them are of high interest to mouse developmental biologists. The node and notochordal plate lie on the ventral surface of gastrulating mouse embryos around embryonic day (E) 7.5 of development. The node is a cup-shaped structure whose cells possess a single slender cilium each. The proper subcellular localization and rotation of the cilia in the node pit determines left-right asymmetry. The notochordal plate cells also possess single cilia albeit shorter than those of the node cells. The notochordal plate forms the notochord which acts as an important signaling organizer for somitogenesis and neural patterning. Because the cells of the node and notochordal plate are transiently present on the surface and possess cilia, they can be visualized using scanning electron microscopy (SEM). Among other techniques used to visualize these structures at the cellular level is whole mount immunofluorescence (WMIF) using the antibodies against the proteins that are highly expressed in the node and notochordal plate. In this report, we describe our optimized protocols to perform SEM and WMIF of the node and notochordal plate in developing mouse embryos to help in the assessment of tissue shape and cellular organization in wild-type and gastrulation mutant embryos.

The node and notochordal plate are transient signaling organizers in developing mouse embryos that can be visualized using several techniques. Here, we describe in detail how to perform two of the techniques to study their structure and morphogenesis: 1) scanning electron microscopy (SEM); and 2) whole mount immunofluorescence (WMIF).

用扫描电镜和全贴免疫荧光技术可视化小鼠胚胎中的节点和外型板

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is ...

Recapitulating Suckling-to-Weaning Transition In Vitro using Fetal Intestinal Organoids

At the end of the suckling period, many mammalian species undergo major changes in the intestinal epithelium that are associated with the capability to digest solid food. This process is termed suckling-to-weaning transition and results in the replacement of neonatal epithelium with adult epithelium which goes hand in hand with metabolic and morphological adjustments. These complex developmental changes are the result of a genetic program that is intrinsic to the intestinal epithelial cells but can, to some extent, be modulated by extrinsic factors. Prolonged culture of mouse primary intestinal epithelial cells from late fetal period, recapitulates suckling-to-weaning transition in vitro. Here, we describe a detailed protocol for mouse fetal intestinal organoid culture best suited to model this process in vitro. We describe several useful assays designed to monitor the change of intestinal functions associated with suckling-to-weaning transition over time. Additionally, we include an example of an extrinsic factor that is capable to affect suckling-to-weaning transition in vivo, as a representation of modulating the timing of suckling-to-weaning transition in vitro. This in vitro approach can be used to study molecular mechanisms of the suckling-to-weaning transition as well as modulators of this process. Importantly, with respect to animal ethics in research, replacing in vivo models by this in vitro model contributes to refinement of animal experiments and possibly to a reduction in the use of animals to study gut maturation processes.

This protocol describes how to mimic suckling-to-weaning transition in vitro using mouse late fetal intestinal organoids cultured for 30 days.

使用胎儿肠道有机体在体外重述吸吮到发胶过渡

At the end of the suckling period, many mammalian species undergo major changes in the intestinal epithelium that are associated with the capability to ...

免疫荧光标记的微管和中心蛋白在体内肠道组织和 3D体外肠道 Organoids

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病毒介导的标签和内侧神经节隆起移植（MGE）细胞

胸腺表征祖定居在小鼠胚胎使用