Name: histological-based stainings using free-floating tissue sections
Uploaded: 2020-08-25T14:30:00
Description: Watch this Scientific Journal Video about Histological-Based Stainings Using Free-Floating Tissue Sections at JoVE.com

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?...

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	<li>Childs, G. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathobiology+of+Human+Disease.+Part+IV:+Techniques+in+Experimental+Pathology.">Pathobiology of Human Disease. Part IV: Techniques in Experimental Pathology.</a> Journal of Experimental Medicine. 381923, 3775-3796 (2014).</li><li>Coons, A. H., Creech, H. J., Jones, R. 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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 ...

This free floating technique can be used to perform for lessened immunohistochemistry on mouse brain tissue samples. The free floating technique provides several advantages. The acquisition of thicker tissue sections, the performance of large scale studies, using 30 to 40 slices per aliquot and the long-term storage of samples.If you are trying this technique for the first time, we recommend you practice mounting sections that are not critical to your experiment to obtain experience with the method. At least one to two hours before sectioning, acclimatize the samples of interest in the cryostat at the appropriate tissue cutting temperature. When the tissues are ready.use the cryostat to acquire 20 to 50 micron thick sections and place the sections into individual inserts in PBS filled wells of a six or 12 well plate. When all of the sections have been acquired, cover the samples with approximately six milliliters per wash for three five minute washes before transferring each well of sections into two milliliter micro centrifuge tubes, containing one to 1.5 milliliters of storage solution per tube. Then store the samples at minus 80 degrees Celsius until staining.For tissue section staining, equilibrate the samples to room temperature for 10 to 20 minutes before decanting the sections into inserts in a new six well plate. When all of the storage solution has been drained, move the inserts into new wells, containing approximately six milliliters of TBS per well for three washes for five minutes with fresh TBS per wash on an orbital shaker at low speed at room temperature. After the last wash, add seven milliliters of freshly prepared blocking permeabilizing solution to each well for a 30 minute incubation on the shaker at room temperature.At the end of the incubation, add one milliliter of the primary antibody solution of interest to one two milliliter micro centrifuge tube per insert and transfer all of the sections in each insert into the appropriate two milliliter tube. Then place the tubes on a rotating mixer. Add about seven revolutions per minute for an overnight incubation at four degrees Celsius.The next morning, decant the sections from each tube into individual well inserts in a new six well plate and wash the sections two times for 30 seconds per wash and one time for 10 minutes in fresh TBS per wash. After the last wash, transfer the groups of sections into individual two milliliter micro centrifuge tubes, containing one milliliter of the appropriate secondary antibody solution per tube and incubate the samples for two hours at room temperature on an orbital shaker at low speed, protected from light. At the end of the incubation, decant the sections into inserts in a new six well plate and wash the samples two times for 30 seconds and one time for 15 minutes in fresh TBS per wash, protected from light.After the last wash, decant the sections into a glass rectangular histological chamber, three quarters filled with TBS and submerge a glass slide in the TBS. Using a fine paintbrush and a magnifying lamp if desired, carefully coax the sections toward the slide and gently tap a section onto the slide, making sure there are no wrinkles or folds. When all of the sections have been captured onto a slide, allow the slides to dry for about 10 to 15 minutes at room temperature, protected from light, until the sections are opaque and apply an appropriate aqueous mounting medium to the sections.Use tweezers to place a cover slip onto the medium and use a piece of filter paper to firmly press down on the cover slip to remove any excess medium. The sections can then be imaged, using an appropriate microscope or stored in a dark slide box set four degree Celsius. As observed in these representative images, fluorescent immunohistochemical analysis, using the free floating method can be performed on mouse brain tissue sections to examine glial fibrillar acidic protein expression.This approach is also appropriate for revealing low expression proteins, such as GFP, in a low expressing transgenic mouse brain sample or in non fluorescent histochemical staining protocols, such as cresyl violet. Other peripheral tissues are also amenable to use of this technique with no modifications required as observed for these low level GFP expressing liver tissue samples. The most important thing to remember with the free floating technique is to be gentle with the sections, especially during transfers and when the samples are on the shakers or rotators.In addition to a immunofluorescence staining, this method can be easily modified for other histochemical stains such as hematoxylin and eosin and cresyl violet or for chromogen immunohistochemistry.

Research

JoVE Journal

Biology

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

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

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

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

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

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

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 ...

Micro-Mechanical Characterization of Lung Tissue Using Atomic Force Microscopy

微型机械的肺组织利用原子力显微镜表征

Matrix stiffness strongly influences growth, differentiation and function of adherent cells1-3.  On the macro scale the stiffness of tissues and organs ...

Chromatin Immunoprecipitation (ChIP) using Drosophila tissue

Chromatin Immunoprecipitation ChIP using Drosophila tissue

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 ...

Indirect Immunofluorescence on Frozen Sections of Mouse Mammary Gland

对小鼠乳腺冰冻切片间接免疫荧光

Indirect immunofluorescence is used to detect and locate proteins of interest in a tissue. The protocol presented here describes a complete and simple ...

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

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

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 ...