我们要感谢国家老龄化研究所（K99 / R00 AG055683至JMR），George and Anne Ryan神经科学研究所（EP，GC，JMR），罗德岛大学药学院（EP，GC，JMR）和Konung Gustaf V：s och Drottning Victorias Frimurarestiftelse（JMR）。我们感谢博士生Rebecca Senft，与哈佛医学院遗传学系的Susan Dymecki教授一起培训，向我们介绍了自由浮动方法。图1中使用的一些图像是从“免费使用，共享或修改，甚至商业上”的来源获得的：小鼠和微量离心管（Pixabay），小鼠大脑（Jonas Töle，维基共享资源），低温恒温器和小鼠大脑部分（数据库生命科学中心，维基共享资源），玻璃容器（OpenClipart，FreeSvg.org）和显微镜（Theresa Knott，Open Clip Art Library）。

1. 冷冻切片的组织准备
<ol><li>将固定组织嵌入适当的包埋模具中（见材料表），以使用适当的试样基质（见材料表）创建试样块，并在干冰上冷冻。将试样块储存在-80°C，直到准备切片。 注意：固定组织通常通过灌注成年（约2.5 - 30个月大）雄性或雌性啮齿动物（小鼠或大鼠）34，根据可用的道德许可，用适当的固定剂（例如10%福尔马林）制备，然后在4°C下在同一固定剂中固定组织12小时，用1x PBS洗涤组织三次， 并将组织放入 15% 和 30% 蔗糖中放入 1x PBS 中过夜或直到组织下沉35。研究人员可能会尝试使这种通用协议适应不同的发展阶段。</li>
</ol>2. 冷冻切片
<ol><li>准备切片时，在切片前将样品在低温恒温器中适应至少1-2小时，以防止组织破碎。</li>
	<li>使用低温恒温器将组织切成切片（20-50μm），并收集在填充有1x PBS溶液的6或12孔插入物（参见 材料表）中。 注意：根据切片厚度，要收集的组织数量以及使用的孔插入物数量，每个孔将包含可变数量的切片，范围从大约10到40个孔切片不等。例如，如果将整个大脑切片为40μm，则使用12孔插入物在每个孔中收集大约18-24个切片。此外，20 μm 切片的处理可能有些困难，因此建议使用 40 μm 进行批量染色（参见讨论）。</li>
</ol>3. 存储部分
<ol><li>收集后，用新鲜制备的1x PBS清洗切片5分钟。重复3次。</li>
	<li>将切片转移到装有 1-1.5 mL 储存溶液的 2 mL 微量离心管中（对于 250 mL，混合 70 g 蔗糖、75 mL 乙二醇，并用 0.1 M 磷酸盐缓冲液体积增大）。</li>
	<li>储存在-80°C直至准备染色。</li>
</ol>4. 染色日 I
<ol><li>从冰箱中取出样品并在室温（RT）下平衡10-20分钟。</li>
	<li>将切片倒入 6 孔板的孔插入物中，以将存储溶液与切片分开。</li>
	<li>将孔插入物移至另一个含有约 6 mL 1x TBS 的孔中。 用 1x TBS 洗涤 3 次，每次 5 分钟，在轨道振荡器上使用室温低速洗涤 5 分钟。</li>
	<li>在切片洗涤时，准备 7 mL（每个样品）的封闭-透化溶液，该溶液由 1x TBS 和 0.3% Triton X-100 和 3% 正常血清（例如，正常马血清）组成。在轨道振动台上的室温下，使用低速块部分30分钟。 注意：用血清封闭可防止抗体与组织或非特异性Fc受体的非特异性结合 - 建议使用与二抗种类匹配的血清，但如果不可用，则可以使用来自与一抗宿主动物不同的物种的任何正常血清。洗涤剂Triton X-100通过透化组织来更好地渗透抗体。</li>
	<li>在含有0.3%Triton X-100和1%正常血清的1x TBS中制备每个由选定的一抗（适当稀释）组成的一抗溶液样品1 mL（参见步骤4.4注意）。将孔中的切片插入到含有一抗溶液的 2 mL 微量离心管中，以与目标抗原结合。 注意：可以使用多种一抗（在不同的宿主物种中产生）。</li>
	<li>使用低速（例如，速度7rpm）将2mL微量离心管与切片放在旋转混合器上，并在4°C下孵育过夜12-16小时。</li>
</ol>5. 染色日 II
<ol><li>第二天，将切片倒入 6 孔板的孔插入物中，以将切片与一抗溶液分离。 注意：抗体溶液可以收集和重复使用;加入0.02%（W / V）叠氮化钠以抑制微生物生长。</li>
	<li>在室温下用 1x TBS 洗涤切片 3 次（前 2 次洗涤为 30 秒，最后一次洗涤为 10 分钟）。</li>
	<li>在含有 0.3% Triton X-100 和 1% 正常血清（避光溶液）的 1x TBS 中，每个由适当的二抗（相应地稀释）组成的二抗溶液样品制备 1 mL。 注意：使用偶联二抗进行间接标记可放大信号，并允许对蛋白质靶标进行比色或荧光可视化。</li>
	<li>将切片转移到含有二抗溶液的 2 mL 微量离心管中。在轨道振荡器上用低速（屏蔽溶液避光）在室温下孵育2小时。</li>
	<li>将切片倒入 6 孔板的孔插入物中，以将切片与二抗溶液分离。</li>
	<li>继续将样品避光，在室温下用 1x TBS 洗涤 2 次 30 秒。然后在 1x TBS 中洗涤 15 分钟，如果需要，加入 DAPI （1-0.1 μg/ml）。</li>
</ol>6. 安装
<ol><li>将切片倒入玻璃矩形组织学室中，其中四分之三充满 1x TBS。</li>
	<li>将载玻片浸入 1x TBS 中，然后用细画笔将切片哄骗到载玻片上。</li>
	<li>轻轻敲击幻灯片上的部分，确保没有皱纹或褶皱。</li>
	<li>重复上述步骤，直到所有部分都安装到载玻片上。 注意：例如，当整个大脑在40μm处切片时，收集在12孔插入物中，一个等分试样包含18-24个切片;切片通常安装在 1-2 张载玻片上，但根据研究人员的喜好，每张载玻片也可以安装更少的切片。</li>
</ol>7. 盖玻片
<ol><li>切片干燥到载玻片上后，在室温下约10-15分钟或直到切片看起来不透明（请记住保护载玻片免受光照），应用适当的水性封片介质（硬化或非硬化）。如果使用荧光偶联二抗，则首选抗褪色。 注意：使用硬化封片剂时，荧光质量可能较低，但载玻片的使用寿命应更长。</li>
	<li>使用镊子将盖玻片放在培养基顶部。盖上滤纸并用力按压以去除多余的安装介质。 注意：如果使用非硬化安装座，请在盖玻片的边缘涂上透明的指甲油以密封。</li>
	<li>使用适当的显微镜对切片进行成像。储存在4°C的深色载玻片盒中。 注意：根据研究人员的需要，可以使用各种显微镜对切片进行成像，例如激光扫描共聚焦和倒置或直立宽场荧光，放大倍率（例如，10x，20x，40x）。</li>
</ol>

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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Immunocytochemistry. , 65-77 (2009).</li><li>Grabinski, T. M., Kneynsberg, A., Manfredsson, F. P., Kanaan, N. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+combining+RNAscope+in+situ+hybridization+with+immunohistochemistry+in+thick+free-floating+brain+sections+and+primary+neuronal+cultures.">A method for combining RNAscope in situ hybridization with immunohistochemistry in thick free-floating brain sections and primary neuronal cultures.</a> PloS one. 10 (3), 0120120 (2015).</li><li>Warr, W. B., de Olmos, J. S., Heimer, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Neuroanatomical Tract-Tracing Methods. , 207-262 (1981).</li><li>Alvarez-Buylla, A., Ling, C. Y., Kirn, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cresyl+violet:+A+red+fluorescent+Nissl+stain.">Cresyl violet: A red fluorescent Nissl stain.</a> Journal of Neuroscience Methods. 33 (23), 129-133 (1990).</li></ol>

没有什么可透露的

免疫组织化学（IHC）是一种多功能技术，对于识别组织切片内的蛋白质表达和定位至关重要。该测定法用于整个科学界，以进一步了解组织在正常功能到疾病状态的各个阶段的特征。IHC用于各个领域，从癌症等疾病的临床诊断到临床前研究的初步发现10，36。
最常用于执行IHC的技术是载玻片安装方法，其中切片后切片后立即将切片?...

免疫染色是一种流行的研究实践，始于 130 年前冯·贝林 1890 年发现的血清抗体1.在20世纪初 ，染料附着在抗原上，后来附着在抗体上，作为量化和可视化反应的一种方式1，1941年，Albert Coons开发了第一个荧光抗体标记，这一发现彻底改变了光学显微镜2，3。术语“免疫染色”包括使用该原理开发的许多技术，包括蛋白质印迹、流式细胞术、ELISA、免疫细胞化学和免疫组织化学3，4。蛋白质印迹法检测组织或细胞提取物中是否存在特定蛋白质5。使用凝胶电泳按大小分离蛋白质，转移到膜上，并使用抗体进行探测。该技术指示蛋白质的存在以及存在多少蛋白质;然而，它不会揭示有关蛋白质在细胞或组织内定位的任何信息。另一种方法是免疫细胞化学（ICC），标记细胞内的蛋白质，通常是体外培养的细胞。ICC显示细胞区室内的蛋白质表达和定位6。为了在组织水平上检测和可视化特定蛋白质，使用了免疫组织化学（IHC）。
IHC是研究人员用来靶向组织内特定抗原的方法，利用免疫系统的化学特性7，8。通过产生与酶或荧光染料相连的特异性一抗和二抗，可以在大多数组织中标记和揭示感兴趣的抗原（如Mepham和Britten所述）9。术语“免疫组织化学”本身并未指定用于揭示目标抗原的标记方法;因此，该术语通常与检测技术相结合，以清楚地描述标记方法：显色免疫组织化学（CIH），用于指示二抗何时与酶（例如过氧化物酶）偶联;或荧光 IHC，以指示二抗何时与荧光团偶联，例如异硫氰酸荧光素 （FITC） 或四甲基罗丹明 （TRITC）。IHC的选择性使临床医生和研究人员能够可视化蛋白质在整个组织中的表达和分布，跨越各种健康和疾病状态10。在临床领域，IHC通常用于诊断癌症，以及确定各种类型癌症的差异。IHC还被用于确认体内不同类型的微生物感染，如乙型肝炎或丙型肝炎11。在生物医学研究中，IHC通常用于绘制组织中的蛋白质表达图谱，并且在识别疾病状态下的异常蛋白质方面很重要。例如，神经变性通常伴随着大脑中异常蛋白质的积累，例如阿尔茨海默病中的Αβ斑块和神经原纤维缠结。然后通常开发动物模型来模拟这些病理状态，IHC是研究人员用来定位和量化感兴趣的蛋白质的一种方法10，12，13。反过来，我们可以更多地了解这些疾病的原因以及随之引起的并发症。
执行 IHC 涉及许多步骤。首先，收集感兴趣的组织并准备染色。可以说，大多数研究人员制备固定组织样本，通过循环系统灌注固定剂是最佳的，因为它保留了形态14，15。也可以使用组织样本的固定后，但可能产生的结果可能不理想16。交联固定剂，如甲醛，通过在组织中的蛋白质之间产生化学键来起作用17。然后使用切片机将固定组织切成非常薄的层或切片，许多研究人员更喜欢使用低温恒温器收集冷冻切片。从那里收集组织并直接安装在显微镜载玻片上（载玻片安装方法），或悬浮在溶液中（自由浮动方法），例如磷酸盐缓冲盐水（PBS）18。所使用的收集方法是根据研究人员的需求预先确定的，这两种方法中的每一种都有自己的优点和缺点。
滑块安装方法是迄今为止最常用的方法，其重要优点是可以制备非常薄的切片（10-14μm），这对于研究蛋白质-蛋白质相互作用非常重要。对标本的处理也最少，这减少了对组织结构完整性的潜在损害19。研究人员经常将这种技术用于新鲜冷冻组织（使用干冰，异戊烷等立即冷冻的组织），与固定组织相比，该技术非常脆弱，需要非常小心地防止样品解冻。使用载玻片安装切片的另一个优点是通常不需要大量染色溶液4。因此，研究人员可以使用少量昂贵的抗体或其他化学物质来完成染色。此外，可以在同一张载玻片上安装来自多个不同实验组的切片，这可能是有利的，尤其是在图像采集期间。另一方面，使用载玻片安装切片存在一些缺点，最明显的是组织切片粘附在切片上，从而限制了抗体渗透到切片的一侧，这限制了切片厚度和组织的3D表示。也可能在洗涤过程中，纸巾的边缘和整个切片可能会从载玻片上脱落，从而使整个实验变得无用。此外，当使用载玻片安装方法时，通常必须相对快速地进行IHC，以避免抗原表位20，21的降解，未处理的载玻片通常储存在-20或-80°C，通常盖玻片并水平或储存在载玻片盒中，导致相对较大的存储足迹。最后，如果研究人员必须处理大量载玻片来处理大量组织切片，则载玻片安装技术也可能非常耗时。
由于使用滑动安装方法的一些挑战，一种称为自由浮动方法的修改已成为一种流行的替代方案。这种技术在 1960-70 年代 22，23，24 进入文献，在 1980 年代25，26，27，28，29 中流行起来，现在是一种成熟的方法，涉及在悬浮液中对收集的部分进行染色而不是粘附在载玻片上12，30，31.当组织切片小于20μm时，不建议使用自由浮动方法;然而，根据我们的经验，当要染色较厚（40-50μm）的切片时，这是首选的方法。一个明显的好处是，抗体可以从各个角度穿透自由漂浮的部分，并且由于更有效的洗涤而产生更少的背景染色，所有这些都可以在成像时产生更好的信号传导。此外，切片在处理后安装在载玻片上，从而消除了组织脱落的可能性，并减少了占用低温恒温器的时间。自由浮动方法的劳动强度也低得多，特别是对于大规模的生物医学研究。例如，可以在同一孔中将同一样品中的许多（18-40）切片一起染色，从而节省执行洗涤和抗体孵育步骤的时间。此外，由于使用这种方法可以在每张载玻片上安装更多（12-16）个切片，因此研究人员查看和成像切片通常更方便、更快捷。值得注意的是，在载玻片上安装组织切片的过程中，可以连接和分离切片，直到获得所需的方向。研究人员还经常使用自由浮动方法使用浓度稍低的抗体，并且由于孵育是在微量离心管中进行的，因此可以很容易地收集抗体并用叠氮化钠保存以供重复使用（参见步骤5.1）。另一个优点是，切片可以直接在-80°C下储存在装有冷冻保护剂溶液32的小型微量离心管中，从而最大限度地减少存储空间并最大限度地延长样品33的使用寿命。使用这种技术的缺点是这些部分处理得很多，因此容易损坏。然而，这可以通过使用低振动和旋转速度以及适当培训研究人员如何转移样品并将切片安装到载玻片上来缓解。
综上所述，IHC是临床和生物医学研究领域可视化和定位蛋白质表达的成熟基本工具。自由漂浮的IHC是一种高效，灵活且经济的方法，特别是在进行大规模组织学研究时。在这里，我们为科学界提供了一种可靠的自由浮动荧光IHC方案，可以相应地适用于显色IHC和其他染色，如苏木精和伊红或甲酚紫染色。

<table>
	<tbody>
		<tr>
			<td>12-well plates</td>
			<td>Corning</td>
			<td>3513</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well plates</td>
			<td>Corning</td>
			<td>3516</td>
			<td></td>
		</tr>
		<tr>
			<td>Clear nail polish</td>
			<td>User preference</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Sigma-Aldrich</td>
			<td>D9542</td>
			<td></td>
		</tr>
		<tr>
			<td>Embedding molds</td>
			<td>Thermo Scientific</td>
			<td>1841</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylene glycol</td>
			<td>User preference</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Formalin solution</td>
			<td>Fisher Scientific</td>
			<td>SF98-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Horse serum, heat inactivated</td>
			<td>Gibco</td>
			<td>26050088</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope slide boxes</td>
			<td>Electron Microscopy Services</td>
			<td>71370</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>User preference</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Primary antibody</td>
			<td>User preference</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Rectangular Coverslips</td>
			<td>VWR</td>
			<td>48393-081</td>
			<td>24 x 50 mm</td>
		</tr>
		<tr>
			<td>Rectangular staining dish</td>
			<td>Electron Microscopy Services</td>
			<td>70312</td>
			<td></td>
		</tr>
		<tr>
			<td>Round artist paintbrush #2</td>
			<td>Princeton Select Series</td>
			<td>3750R</td>
			<td>Brand not important</td>
		</tr>
		<tr>
			<td>Secondary antibody</td>
			<td>User preference</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Specimen matrix for embedding</td>
			<td>OCT Tissue-Tek, Sakura</td>
			<td>4583</td>
			<td></td>
		</tr>
		<tr>
			<td>Stain tray &#8211; slide staining system</td>
			<td>Electron Microscopy Services</td>
			<td>71396-B</td>
			<td>Use dark lid</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>User preference</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Superfrost Plus Micro Slides</td>
			<td>VWR</td>
			<td>48311-703</td>
			<td></td>
		</tr>
		<tr>
			<td>TBS</td>
			<td>User preference</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>X100</td>
			<td></td>
		</tr>
		<tr>
			<td>Vectashield antifade mounting medium</td>
			<td>Vector Laboratories</td>
			<td>H-1000</td>
			<td>Non-hardening</td>
		</tr>
		<tr>
			<td>Well inserts for 12-well plates</td>
			<td>Corning Netwells</td>
			<td>3477</td>
			<td></td>
		</tr>
		<tr>
			<td>Well inserts for 6-well plates</td>
			<td>Corning Netwells</td>
			<td>3479</td>
			<td></td>
		</tr>
		<tr>
			<td>Whatman filter paper</td>
			<td>Millapore-Sigma</td>
			<td>WHA1440042</td>
			<td></td>
		</tr>
	</tbody>
</table>

histological-based stainings using free-floating tissue sections

免疫组织化学是一种广泛使用的技术，用于可视化特定组织结构以及蛋白质表达和定位。在染色过程中，两种替代方法广泛用于处理组织切片，一种方法包括将切片直接安装在载玻片上，而第二种方法，自由浮动，允许在悬浮在溶液中时保持固定切片并进行染色。尽管载玻片安装和自由浮动方法可能会产生类似的结果，但自由浮动技术可以更好地穿透抗体，因此当较厚的切片用于组织的3D重建时，例如当实验的重点是获得有关大脑区域的树突状和轴突投影的信息时，应该是首选方法。此外，由于切片保存在溶液中，因此单个等分试样可以轻松容纳 30 到 40 个切片，处理这些切片的工作量较小，尤其是在大规模生物医学研究中。在这里，我们说明了如何将自由浮动方法应用于荧光免疫组化染色，主要关注脑切片。我们还将讨论如何轻松修改自由浮动技术以满足研究人员的个性化需求，并适应其他组织以及其他基于组织化学的染色，如苏木精和伊红和甲酚紫，只要组织样品正确固定，通常用多聚甲醛或福尔马林。

使用自由浮动法进行荧光免疫组织化学测定的总体方案如图 1所示。使用自由浮动方法在小鼠脑中检查神经胶质原纤维酸性蛋白（GFAP）表达的荧光IHC的代表性示例如图 2 所示，在较低和较高的放大倍率下以说明染色的整体质量。这种方法也适用于揭示低表达蛋白， 图3所示为GFP低表达转基因小鼠大脑的示例。自由浮动方法也可用?...

Watch this Scientific Journal Video about Histological-Based Stainings Using Free-Floating Tissue Sections at JoVE.com

Histological-Based Stainings Using Free-Floating Tissue Sections

自由浮动技术允许研究人员进行基于组织学的染色，包括对固定组织切片进行免疫组织化学，以可视化生物结构、细胞类型以及蛋白质表达和定位。这是一种高效可靠的组织化学技术，可用于研究多种组织，例如大脑、心脏和肝脏。

使用自由浮动组织切片进行基于组织学的染色

Immunohistochemistry is a widely used technique to visualize specific tissue structures as well as protein expression and localization. Two alternative ...

histological-based-stainings-using-free-floating-tissue-sections

Research

JoVE Journal

Biology

17.6K 次观看.  University of Rhode Island. 自由浮动技术允许研究人员进行基于组织学的染色，包括对固定组织切片进行免疫组织化学，以可视化生物结构、细胞类型以及蛋白质表达和定位。这是一种高效可靠的组织化学技术，可用于研究多种组织，例如大脑、心脏和肝脏。

视频: Histological-Based Stainings Using Free-Floating Tissue Sections

Immunohistochemistry: Paraffin Sections Using the Vectastain ABC Kit from Vector Labs

Immunohistochemistry (IHC) is a valuable technique utilized to localize/visualize protein expression in a mounted tissue section using specific antibodies. There are two methods: the direct and indirect method. In this experiment, we will only describe the use of indirect IHC staining. Indirect IHC staining utilizes highly specific primary and biotin-conjugated secondary antibodies. Primary antibodies are utilized to discretely identify proteins of interest by binding to a specific epitope, while secondary antibodies subtract for non-specific background staining and amplify signal by forming complexes to the primary antibody. Slides can either be generated from frozen sections, or paraffin embedded sections mounted on glass slides. In this protocol, we discuss the preparation of paraffin-embedded sections by dewaxing, hydration using an alcohol gradient, heat induced antigen retrieval, and blocking of endogenous peroxidase activity and non-specific binding sites. Some sections are then stained with antibodies specific for T cell marker CD8 and while others are stained for tyrosine hydroxylase. The slides are subsequently treated with appropriate secondary antibodies conjugated to biotin, then developed utilizing avidin-conjugated horseradish peroxidase (HRP) with Diaminiobenzidine (DAB) as substrate. Following development, the slides are counterstained for contrast, and mounted under coverslips with permount. After adequate drying, these slides are then ready for imaging.

免疫组化：石蜡切片从Vector实验室使用Vectastain ABC试剂盒

Immunohistochemistry (IHC) is a valuable technique utilized to localize/visualize protein expression in a mounted tissue section using specific ...

科研

生物学

Immunohistochemistry on Paraffin Sections of Mouse Epidermis Using Fluorescent Antibodies

In the epidermis, immunohistochemistry is an efficient means of localizing specific proteins to their relative expression compartment; namely the basal, suprabasal, and stratum corneum layers. The precise localization within the epidermis of a particular protein lends clues toward its functional role within the epidermis. In this chapter, we describe a reliable method for immunolocalization within the epidermis modified for both frozen and paraffin sections that we use very routinely in our laboratory. Paraffin sections generally provide much better morphology, hence, superior results and photographs; however, not all antibodies will work with the harsh fixation and treatment involved in their processing. Therefore, the protocol for frozen sectioning is also included. Within paraffin sectioning, two fixation protocols are described (Bouin's and paraformaldehyde); the choice of fixative will be directly related to the antibody specifications and may require another fixing method.

We describe a reliable method for immunolocalization within the epidermis modified for both frozen and paraffin sections that we use very routinely in our laboratory.

使用荧光抗体对小鼠表皮石蜡切片的免疫组织化学

In the epidermis, immunohistochemistry is an efficient means of localizing specific proteins to their relative expression compartment; namely the basal, ...

Rapid Genotyping of Mouse Tissue Using Sigma's Extract-N-Amp Tissue PCR Kit

Genomic detection of DNA via PCR amplification and detection on an electrophoretic gel is a standard way that the genotype of a tissue sample is determined. Conventional preparation of tissues for PCR-ready DNA often take several hours to days, depending on the tissue sample. The genotype of the sample may thus be delayed for several days, which is not an option for many different types of experiments. Here we demonstrate the complete genotyping of a mouse tail sample, including tissue digestion and PCR readout, in one and a half hours using Sigma's SYBR Green Extract-N-Amp Tissue PCR Kit. First, we demonstrate the fifteen-minute extraction of DNA from the tissue sample. Then, we demonstrate the real time read-out of the PCR amplification of the sample, which allows for the identification of a positive sample as it is being amplified. Together, the rapid extraction and real-time readout allow for a prompt identification of genotype of a variety different types of tissues through the reliable method of PCR.

Rapid Genotyping of Mouse Tissue Using Sigma&#039;s Extract-N-Amp Tissue PCR Kit

The complete genotyping of a mouse tail sample, including tissue digestion and PCR readout, is done in one and a half hours using Sigma's SYBR Green Extract-N-Amp Tissue PCR kit.

使用Sigma的提取物N -安培组织PCR试剂盒小鼠组织的快速基因分型

Genomic detection of DNA via PCR amplification and detection on an electrophoretic gel is a standard way that the genotype of a tissue sample is ...

Actin Co-Sedimentation Assay; for the Analysis of Protein Binding to F-Actin

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates tension and helps maintains cellular shape.  Although the actin cytoskeleton is a rigid structure, it is a dynamic structure that is constantly remodeling.  A number of proteins can bind to the actin cytoskeleton.  The binding of a particular protein to F-actin is often desired to support cell biological observations or to further understand dynamic processes due to remodeling of the actin cytoskeleton. The actin co-sedimentation assay is an in vitro assay routinely used to analyze the binding of specific proteins or protein domains with F-actin.  The basic principles of the assay involve an incubation of the protein of interest (full length or domain of) with F-actin, ultracentrifugation step to pellet F-actin and analysis of the protein co-sedimenting with F-actin.  Actin co-sedimentation assays can be designed accordingly to measure actin binding affinities and in competition assays.

Proteins bind to filamentous actin (F-actin) through distinct actin binding modules. In this video we demonstrate the procedure of actin co-sedimentation, which is an in vitro assay routinely used to analyze proteins or specific domains that bind F-actin.

肌动蛋白共沉淀测定;​​ F -肌动蛋白结合蛋白分析

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates ...

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of muscle specification, formation and function in model systems has provided valuable insight into muscle physiology. Therefore, identifying and characterizing molecular mechanisms that underlie muscle damage is critical. The structure of adult Drosophila multi-fiber muscles resemble vertebrate striated muscles 1 and the genetic tractability of Drosophila has made it a great system to analyze dystrophic muscle morphology and characterize the processes affecting muscular function in ageing adult flies 2. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. These preparations allow for the tissue to be stained with classical histological stains and labeled with protein detecting dyes, and specifically cryosections are ideal for immunohistochemical detection of proteins in intact muscles. This allows for analysis of muscle tissue structure, identification of morphological defects, and detection of the expression pattern for muscle/neuron-specific proteins in Drosophila adult muscles. These techniques can also be slightly modified for sectioning of other body parts.

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

Identification of mechanisms underlying muscle damage is crucial. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. This allows analysis of muscle morphology and localization of protein and other muscle cell components.

石蜡包埋及冷冻的部分果蝇成人肌肉

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of ...

Chromatin Immunoprecipitation (ChIP) using Drosophila tissue

Epigenetics remains a rapidly developing field that studies how the chromatin state contributes to differential gene expression in distinct cell types at different developmental stages. Epigenetic regulation contributes to a broad spectrum of biological processes, including cellular differentiation during embryonic development and homeostasis in adulthood. A critical strategy in epigenetic studies is to examine how various histone modifications and chromatin factors regulate gene expression. To address this, Chromatin Immunoprecipitation (ChIP) is used widely to obtain a snapshot of the association of particular factors with DNA in the cells of interest.
ChIP technique commonly uses cultured cells as starting material, which can be obtained in abundance and homogeneity to generate reproducible data. However, there are several caveats: First, the environment to grow cells in Petri dish is different from that in vivo, thus may not reflect the endogenous chromatin state of cells in a living organism. Second, not all types of cells can be cultured ex vivo. There are only a limited number of cell lines, from which people can obtain enough material for ChIP assay.
Here we describe a method to do ChIP experiment using Drosophila tissues. The starting material is dissected tissue from a living animal, thus can accurately reflect the endogenous chromatin state. The adaptability of this method with many different types of tissue will allow researchers to address a lot more biologically relevant questions regarding epigenetic regulation in vivo1, 2. Combining this method with high-throughput sequencing (ChIP-seq) will further allow researchers to obtain an epigenomic landscape.

Chromatin Immunoprecipitation ChIP using Drosophila tissue

Recently high-throughput sequencing technology has greatly increased sensitivity of Chromatin Immunoprecipitation (ChIP) experiment and prompted its application using purified cells or dissected tissue. Here we delineate a method to use ChIP technique with Drosophila tissue, which can address the endogenous chromatin state in a well-characterized biological system.

染色质免疫沉淀（ChIP）使用果蝇组织

Epigenetics remains a rapidly developing field that studies how the chromatin state contributes to differential gene expression in distinct cell types at ...

A Rapid Automated Protocol for Muscle Fiber Population Analysis in Rat Muscle Cross Sections Using Myosin Heavy Chain Immunohistochemistry

Quantification of muscle fiber populations provides a deeper insight into the effects of disease, trauma, and various other influences on skeletal muscle composition. Various time-consuming methods have traditionally been used to study fiber populations in many fields of research. However, recently developed immunohistochemical methods based on myosin heavy chain protein expression provide a quick alternative to identify multiple fiber types in a single section. Here, we present a rapid, reliable and reproducible protocol for improved staining quality, allowing automatic acquisition of whole cross sections and automatic quantification of fiber populations with ImageJ. For this purpose, embedded skeletal muscles are cut in cross sections, stained using myosin heavy chains antibodies with secondary fluorescent antibodies and DAPI for cell nuclei staining. Whole cross sections are then scanned automatically using a slide scanner to obtain high-resolution composite pictures of the entire specimen. Fiber population analyses are subsequently performed to quantify slow, intermediate and fast fibers using an automated macro for ImageJ. We have previously shown that this method can identify fiber populations reliably to a degree of &#177;4%. In addition, this method reduces inter-user variability and time per analyses significantly using the open source platform ImageJ.

Here, we present a protocol for rapid muscle fiber analyses, which allows improved staining quality, and thereby automatic acquisition and quantification of fiber populations using the freely available software ImageJ.

在使用肌球蛋白重链免疫鼠肌肉横断面的快速自动化方案为肌纤维人口分析

Quantification of muscle fiber populations provides a deeper insight into the effects of disease, trauma, and various other influences on skeletal muscle ...

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

The actin binding protein girdin is a cytosolic protein that is required for actin remodeling to trigger cell migration in various tissues. Girdin is phosphorylated by both receptor and non-receptor tyrosine kinases at tyrosine 1798. Omori et al. developed site- and phosphorylation status-specific antibodies against human girdin at tyrosine-1798 (pY1798), which specifically bind to phosphorylated tyrosine-1798, but not to unphosphorylated tyrosine-1798. pY1798 antibodies have been used to specifically label tuft cells (TCs) that are present in mammalian gastrointestinal tissues, but the function of these cells is unclear. This protocol allows the robust visualization of TCs in the jejunum using pY1798 antibodies and immunofluorescence. To ensure successful and simple TC visualization, this protocol includes two histological techniques: production of free-floating cryosections from gelatin-filled jejunum tissue, and low-temperature antigen retrieval at 50 &#176;C for 3 h. Filling the jejunum with gelatin maintains the shape of free-floating sections throughout the staining procedure, whereas low-temperature antigen retrieval ensures robust signals from TCs. Successful use of this protocol results in pY1798 staining of TCs distributed from villus tip to crypt. Stained TCs have a spool-shaped soma and fluorescent signals condense at the lumenal tip, which corresponds to the protruding &#39;tuft.&#39;&#160;Phalloidin staining colocalized with pY1798-positive TCs at the thickened brush border, and corresponds to a rootlet mass extending from the TC tuft. This protocol could be used to examine TCs in human biopsy samples collected with gastrointestinal endoscopes. Furthermore, TCs were recently reported to accumulate following parasite infection in mice, suggesting that this protocol could have applications for diagnosis of parasite infections in the human gut.

Kuga et al. discovered that phosphorylation-status specific antibodies against the actin binding protein girdin phosphorylated at tyrosine 1798 (pY1798) can be used to label tuft cells (TCs). This protocol allows robust visualization of TCs using immunofluorescent staining of free-floating jejunum cryosections with pY1798 antibodies.

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

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

Pancreatic Islet Embedding for Paraffin Sections

Experiments using isolated pancreatic islets are important for diabetes research, but islets are expensive and of limited abundance. Islets contain a mixed cell population in a structured architecture that impacts function, and human islets are widely variable in cell type composition. Current frequently used methods to study cultured islets include molecular studies performed on whole islets, lumping disparate islet cell types together, or microscopy or molecular studies on dispersed islet cells, disrupting islet architecture. For in vivo islet studies, paraffin-embedded pancreas sectioning is a powerful technique to assess cell-specific outcomes in the native pancreatic environment. Studying post-culture islets by paraffin sectioning would offer several advantages: detection of multiple outcomes on the same islets (potentially even the exact-same islets, using serial sections), cell-type-specific measurements, and maintaining native islet cell-cell and cell-substratum interactions both during experimental exposure and for analysis. However, existing techniques for embedding isolated islets post-culture are inefficient, time consuming, prone to loss of material, and generally produce sections with inadequate islet numbers to be useful for quantifying outcomes. Clinical pathology laboratory cell block preparation facilities are inaccessible and impractical for basic research laboratories. We have developed an improved, simplified bench-top method that generates sections with robust yield and distribution of islets. Fixed islets are resuspended in warm histological agarose gel and pipetted into a flat disc on a standard glass slide, such that the islets are distributed in a plane. After standard dehydration and embedding, multiple (10+) 4 - 5 &#181;m sections can be cut from the same islet block. Using this method, histological&#160;and immunofluorescent analyses can be performed on mouse, rat, and human islets. This is an effective, inexpensive, time-saving approach to assess cell-type-specific, intact-architecture outcomes from cultured islets.

Ex vivo pancreatic islet studies are important for diabetes research. Existing techniques to study cultured islets in their native 3-dimensional architecture are time consuming, inefficient, and infrequently used. This work describes a new, simple, and efficient method for generating high-quality paraffin sections of whole cultured islets.

胰岛嵌段石蜡切片

Experiments using isolated pancreatic islets are important for diabetes research, but islets are expensive and of limited abundance. Islets contain a ...

Y-27632 Enriches the Yield of Human Melanocytes from Adult Skin Tissues

The isolation and culture of primary melanocytes from skin tissues is very important for biological research and has been widely used for clinical applications. Isolating primary melanocytes from skin tissues by the conventional method usually takes about 3 to 4 weeks to passage sufficiently. More importantly, the tissues used are usually newborn foreskins and it is still a challenge to efficiently isolate primary melanocytes from adult tissues. We recently developed a new isolation method for melanocytes that adds Y-27632, a Rho kinase inhibitor, to the initial culture medium for 48 h. Compared with the conventional protocol, this new method dramatically increases the yield of melanocytes and shortens the time required to isolate melanocytes from foreskin tissues. We now describe this new method in more detail using adult epidermis to efficiently culture primary melanocytes. Importantly, we show that melanocytes obtained from adult tissues prepared by this new method can function normally. This new protocol will significantly benefit studies of pigmentation defects and melanomas using primary melanocytes prepared from easily accessed adult skin tissues.

This paper reports that the addition of Y-27632 to TIVA medium can significantly increase the yield of melanocytes from adult skin tissues.