Name: tissue preparation and immunostaining of mouse craniofacial tissues and undecalcified bone
Uploaded: 2019-05-10T13:00:00
Description: Watch this Scientific Journal Video about Tissue Preparation and Immunostaining of Mouse Craniofacial Tissues and Undecalcified Bone at JoVE.com

这项工作得到了国家卫生研究院(R01DE020843至Y.M.)、国际FOP协会(Y.M.)和中国国家自然科学基金资助(31500788至J.Y.)的支持。

所有小鼠实验都按照密歇根大学关于人道护理和动物在研究中使用的指导方针进行。这项研究中使用的所有动物程序都通过了密歇根大学(协议#PRO00007715)的机构动物护理和使用委员会(IACUC)。
1. 组织准备
<ol><li>
胚胎组织制剂
	<ol>
<li>为每个怀孕的小鼠准备一个 10 厘米的盘子和几个含有磷酸盐缓冲盐水 (PBS) 的 3.5 厘米菜,以及一个含有 2 mL 4% 甲醛 (PFA) 的 12 孔培养板...

<ol>
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在这里,我们提供了一个详细的协议,用于制备小鼠头和未去切骨组织,以及用于细胞增殖、细胞死亡和BMP信号标记的免疫染色的冷冻切片。我们还详细介绍了从免疫荧光图像获取定量数据的策略。这些方法也适用于具有适当修改的其他组织。
组织制备的条件因组织的大小和类型而异。固定和冷冻保护时间通常需要几个小时到一夜之间。固定后,组织也可以嵌入石蜡和切片与微托<...

面部是人类身份的关键部分,由几种不同的组织组成,如上皮、肌肉、骨骼、软骨、牙齿。这些组织来自所有三个生殖层、内皮和中代1、2。为了正确形成颅面组织,细胞增殖、死亡和分化需要高度协调和由特定的信号通路调节,如Wnt、Fgf、Hh和Bmp通路3、4 , 5.细胞增殖、存活或分化的缺陷会导致颅面畸形,这是最常见的先天性先天缺陷之一。转基因小鼠是研究颅面形态形成和发病机制的有用工具,其作用是1、2、3、4、5。了解颅面结构在发育和发病过程中的变化将有助于澄清关键的发育原理以及颅面畸形的机制1,2,3 ,4,5.
用特定抗体染色整个安装或切片组织是确定感兴趣的蛋白质的空间分布的宝贵技术。从形式上讲,组织免疫染色可以依赖于免疫组织化学 (IHC) 或免疫荧光 (IF)。与IHC用3,3'-Diaminobenzidine(DAB)等致色基质产生的不透明反应产物相比,IF涉及使用荧光显微镜可见的荧光结合物。因此,IF 可以清楚地区分正细胞和背景噪声,并允许通过 ImageJ 和 Adobe Photoshop7、8等软件以简单的方式对图像进行定量分析和增强。整个安装染色方法适用于小块组织(厚度小于 5 mm),可提供蛋白质/抗原位置的三维信息,而无需从第9节、第 10 节开始重建.然而,与组织部分相比,整个安装免疫染色是耗时的,需要大量的抗体溶液。并非所有抗体都与基本的整体安装方法兼容。此外,抗体的不完全渗透将导致不均匀的染色或错误的阴性染色。在这里,我们将重点介绍切片组织上蛋白质/抗原的免疫荧光检测。对于硬组织(如头部、牙齿、长骨),钙在发育/发病过程中沉积使样品难以分段,在免疫染色治疗期间容易冲洗掉11、12。目前可用的大多数协议在嵌入之前对硬组织进行标记,使切片更容易,这非常耗时,如果处理不当,可以破坏样品的形态和抗原。为了克服这些问题,我们优化了一种在不去钙化的情况下冷冻硬组织的方法,从而改进了信号蛋白的形态和分布的可视化。
此处描述的方案用于确定BMP转基因小鼠颅面组织的形态学和组织学变化。具体来说,该方案包括(1)收获和解剖头组织,(2)实验标记的截面和免疫染色(Ki67,pSmad1/5/9)以及TUNEL染色,(3)使用荧光显微镜成像部分,最后(4)分析和量化结果。准备和冷冻硬组织没有去钙化的协议也描述了13。这些方法针对颅面组织进行了优化。它们也适用于不同年龄的样品的其他组织,并作适当的修改。

<table border="0" cellpadding="0" cellspacing="0" style="width:752px;" width="752">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Adhesive tape</td>
			<td style="width:171px;">Leica</td>
			<td style="width:119px;">#39475214</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:296px;">Alexa fluor 488-goat anti-Rabbit secondary antibody</td>
			<td style="width:171px;">Invitrogen</td>
			<td style="width:119px;">A-11034</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Antifade Mountant with DAPI</td>
			<td style="width:171px;">Invitrogen</td>
			<td style="width:119px;">P36931</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Bovine serum albumin</td>
			<td style="width:171px;">Sigma</td>
			<td style="width:119px;">A2153</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Coverslips</td>
			<td style="width:171px;">Fisher Brand</td>
			<td style="width:119px;">12-545-E</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Cryostat</td>
			<td style="width:171px;">Leica</td>
			<td style="width:119px;">CM1850</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">EDTA</td>
			<td style="width:171px;">Sigma</td>
			<td style="width:119px;">E6758</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Fluorescence microscope</td>
			<td style="width:171px;">Olympus</td>
			<td style="width:119px;">BX51</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Gelatin</td>
			<td style="width:171px;">Sigma</td>
			<td style="width:119px;">G1890</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">In Situ&#160;Cell Death Detection Kit</td>
			<td style="width:171px;">Millipore</td>
			<td style="width:119px;">S7165</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Microscope slides</td>
			<td style="width:171px;">Fisher Brand</td>
			<td style="width:119px;">12-550-15</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">OCT Compound</td>
			<td style="width:171px;">Fisher Healthcare</td>
			<td style="width:119px;">23-730-571</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Paraformaldehyde (PFA)</td>
			<td style="width:171px;">Sigma</td>
			<td style="width:119px;">P6148&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Phosphate buffered saline (PBS)</td>
			<td style="width:171px;">Sigma</td>
			<td style="width:119px;">P4417</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Polyethylene glycol&#160;tert-octylphenyl ether</td>
			<td style="width:171px;">Sigma</td>
			<td style="width:119px;">T9284</td>
			<td style="width:167px;">Triton X-100</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Proteinase K</td>
			<td style="width:171px;">Invitrogen</td>
			<td style="width:119px;">AM2542</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:296px;">Rabbit anti-Ki67 antibody</td>
			<td style="width:171px;">Cell Signaling Technology</td>
			<td style="width:119px;">9129</td>
			<td>Lot#:3; RRID:AB_2687446</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:296px;">Rabbit anti-pSmad1/5/9 antibody</td>
			<td style="width:171px;">Cell Signaling Technology</td>
			<td style="width:119px;">13820</td>
			<td>Lot#:3; RRID:AB_2493181</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Sodium citrate</td>
			<td style="width:171px;">Sigma</td>
			<td style="width:119px;">1613859</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Sucrose</td>
			<td style="width:171px;">Sigma</td>
			<td style="width:119px;">S9378</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Tris</td>
			<td style="width:171px;">Sigma</td>
			<td style="width:119px;">10708976001</td>
			<td></td>
		</tr>
	</tbody>
</table>

tissue preparation and immunostaining of mouse craniofacial tissues and undecalcified bone

组织免疫染色提供对给定组织内感兴趣的蛋白质的高度特定和可靠的检测。在这里,我们描述了一个完整和简单的协议,以小鼠颅面组织为例,用于检测颅面形态形成/发病过程中的蛋白质表达。该方案包括组织制备和冷冻、间接免疫荧光、图像采集和定量。此外,还介绍了一种制备和冷冻未分化的硬组织进行免疫染色的方法,以颅面组织和长骨为例。这些方法是确定颅面形态形成/发病过程中各种组织中的蛋白质表达和形态/解剖变化的关键。它们也适用于具有适当修改的其他组织。组织学知识和各节的高质量对于从实验结果中得出科学结论至关重要。该方法的潜在局限性包括但不限于抗体的特异性和定量困难,这里还讨论了这些缺陷。

胚胎颅面组织部分 按照上述步骤,在胚胎日(E)16.5或18.5时,从对照(P0-Cre)或突变体(神经峰细胞中形成激活的Bmpr1a,P0-Cre;caBmpr1a)胚胎中解剖头部。 在4%PFA中固定4小时后,样品嵌入OCT和低温分度。结果部分被免疫染色的抗体对pSmad1/5/9(下游BMP信号因子)或Ki67(细胞增殖标记)没有抗原检索根据协议。如图所示,pSmad1/5/9(图1A)和Ki67(<strong c...

Watch this Scientific Journal Video about Tissue Preparation and Immunostaining of Mouse Craniofacial Tissues and Undecalcified Bone at JoVE.com

Tissue Preparation and Immunostaining of Mouse Craniofacial Tissues and Undecalcified Bone

小鼠颅面组织和未切除骨骼的组织制备及免疫染色

在这里,我们提出了一个详细的协议,以小鼠颅面组织为例,通过免疫染色来检测和量化颅面形态形成/发病机制期间的蛋白质水平。此外,我们描述了一种制备和冷冻方法,从幼鼠的未分化硬组织免疫染色。

Tissue immunostaining provides highly specific and reliable detection of proteins of interest within a given tissue. Here we describe a complete and ...

在这里，我们提出了我们简单的协议，用于在分节材料上的蛋白质上进行免疫荧光检测。此方法不需要抗原检索。我们将使用鼠标头，包括未计算硬组织。该技术的主要优点是跳过硬组织抗原检索和脱钙。这些导致免疫污染效果的质量良好。这种方法还节省了时间和人工。此方法也适用于其他组织，并适当修改。为了从未计算的组织中制作好部分，进一步解剖目标组织以修剪周围的组织，如有必要，在低温保护前两天，在四摄氏度下用 10%EDTA 处理样品。可视化演示对该方法的过程进行了详细而清晰的说明。首先，为每只怀孕小鼠准备一个10厘米和几个3.5厘米的含有PBS的盘子和一个含有两毫升4%PFA的培养板，每个井内都有4%PFA的培养板，并把它们全部放在冰上。要解剖怀孕小鼠的胚胎，用钳子抓住安乐死小鼠腹部中心下方的皮肤，只切开皮肤，然后轻轻拉皮，将其从下腹部肌肉壁分离。按照相同的皮肤切口线，切入腹腔。取出含有一串胚胎的子宫。然后，通过轻轻切开子宫壁来去除胚胎，子宫壁将去除外质组织，如蛋黄囊和安尼翁。切割并分离头部，从每个胚胎，用钳子将每个头部转移到一个包含4%PFA的12井板的单独井中。在四摄氏度下孵育四小时以修复。在 4 摄氏度的 PBS 中冲洗头部，轻轻摇动 12 小时。要冷冻保护头部，请用钳子将其转移到新的 12 井板中，其中含有 PBS 中 30%蔗糖的两毫升。在四摄氏度下轻轻的搅拌孵育，直到头部沉入菜底。要嵌入低温保护头，请将它们转移到含有最佳切割温度化合物的模具中，并平衡几分钟。使用钳子，调整样品的位置和方向，使样品的修剪侧朝向嵌入模具的底部。然后，将模具放在干冰上进行冷冻，并存放在零下80摄氏度的塑料袋中，直到准备进行冷冻。对于产后组织，在切除安乐死三周至三个月大的小鼠的皮肤和脂肪组织后，切开并隔离头部，取出粘结物。然后，修复和冷冻保护头部，就像用胚胎头做。以与 OCT 中的胚胎头类似的方式嵌入 8% 明胶中，并将 Cryomolds 放在零下 80 摄氏度的塑料袋中，直到冷冻。设置适当的低温恒温。将样品放在低温室中约 30 分钟，以与低温恒温均衡。将带样品的块从 Cryomold 中拆下，然后用 OCT 滴将其安装到试样夹头上并冻结，确保样品的修剪侧远离夹头并朝向操作员。将块安装夹头装载到低温器对象支架上。调整刀片支架，使铲刀角度相对于样品保持 3 到 5 度。将 10 微米部分收集到涂层显微镜幻灯片上。将部分留在室温下，直到完全干燥，然后将它们储存在零下80摄氏度。首先进行染色，从零下80摄氏度的零下80摄氏度中拿出滑梯，并在室温下保持一小时，以空气干燥部分。在0.1%PBST中冲洗幻灯片三次，每次五分钟，洗净十进制，并渗透部分。在每个幻灯片中加入 200 微升的阻消溶液，并在室温下孵育 30 分钟。然后，在不冲洗幻灯片的情况下移除阻塞解决方案。在每张幻灯片中加入100微升的原抗体稀释，并在4摄氏度下孵育。在室温下，用 PBS 冲洗幻灯片三次，每次冲洗 10 分钟。加入100微升的二次抗体稀释在阻消溶液中，并在室温下孵育一小时，免受光线影响。在室温下用PBS冲洗三次，每次冲洗10分钟，仍然防止光线。要安装幻灯片，请在每张幻灯片上添加两滴带 DAPI 的防褪色介质，盖上盖玻片，并在 4 摄氏度下存储，直到准备好成像。控制胚胎的正面骨骼对pSmad1/5/9、下游BMP信号因子和细胞增殖标记Ki67呈阳性。然而，在神经波峰特异性，组成激活BMPR1A突变胚胎，然而，pSmad1/5/9水平增加，而Ki67水平下降。此外，在突变胚胎的正面骨骼中观察到的凋亡细胞比控制胚胎的细胞多。明胶嵌入头部的冠状冷冻清晰显示来自鼻骨和鼻组织tdTomato盒的GFP信号和RFP信号，表明明胶不会干扰荧光信号。当这些部分被免疫污为SOX9或Osterix和E11/波多普兰素，优质部分从大多数三个星期的硬组织获得。另一方面，用三个月大的样本，只在一些硬组织，包括股骨、鼻组织和头骨的血管隔间，包括鼻腔缝合线和头部周围的骨头中，只获得了高质量的部分。SOX9阳性细胞特别在生长板的软骨细胞和股骨和鼻隔膜的关节中检测。在三周大的切口中，SOX9在分血细胞中被发现。奥斯特里克斯和E11双染色结果表明，在骨细胞中检测到奥斯特里克斯，而E11在股骨和头部骨骼的骨细胞中检测到。在三周的切口中，奥斯特利克斯在细胞质中呈阳性，而E11在卵泡中质细胞中呈阳性。请不要用 4%PFA 过度修复样品。对于未计算硬组织在明胶中的分块，最佳温度为零下25摄氏度，更低。按照这个程序，荧光水平可以量化，如我们的协议中描述的。这些测量将提供翔实的数据，以显示不同组之间蛋白质相对水平的差异。在它的发展之后，这项技术为双重或三重染色铺平了道路，以获得不同蛋白质的共表达模式。4%PFA 是危险的。它可能导致皮肤过敏、严重眼损伤、器官损伤或癌症。请使用个人防护设备，处理后彻底清洗皮肤。

Research

JoVE Journal

Developmental Biology

Automated Quantification of Hematopoietic Cell &#8211; Stromal Cell Interactions in Histological Images of Undecalcified Bone

造血干细胞的自动定量 - 在脱钙骨的组织学图像间质细胞的相互作用

Confocal microscopy is the method of choice for the analysis of localization of multiple cell types within complex tissues such as the bone marrow. ...

科研

发育生物学

Generating Primary Fibroblast Cultures from Mouse Ear and Tail Tissues

从鼠耳和尾组织生成主成纤维细胞培养

Primary cells are derived directly from tissue and are thought to be more representative of the physiological state of cells in vivo than established cell ...

Identification of Critical Conditions for Immunostaining in the Pea Aphid Embryos: Increasing Tissue Permeability and Decreasing Background Staining

临界条件在免疫染色豌豆蚜虫胚胎鉴定：增加组织通透性和降低背景染色

The pea aphid Acyrthosiphon pisum, with a sequenced genome and abundant phenotypic plasticity, has become an emerging model for genomic and developmental ...

Live Imaging of Mouse Secondary Palate Fusion

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 Coronal Slice Preparation of Developing Mouse Pituitary Gland

发育小鼠垂体的解剖和冠状切片制备

The pituitary gland or hypophysis is an important endocrine organ secreting hormones essential for homeostasis. It consists of two glands with separate ...

Dissection, Culture and Analysis of Primary Cranial Neural Crest Cells from Mouse for the Study of Neural Crest Cell Delamination and Migration

小鼠原发性颅神经冠细胞的解剖、培养与分析，用于神经冠细胞脱角和迁移研究

Over the past several decades there has been an increased availability of genetically modified mouse models used to mimic human pathologies. However, the ...

Clonal Genetic Tracing using the Confetti Mouse to Study Mineralized Tissues

使用纸屑小鼠研究矿化组织的克隆遗传追踪

Labeling an individual cell in the body to monitor which cell types it can give rise to and track its migration through the organism or determine its ...

Laser Capture Microdissection of Mouse Embryonic Cartilage and Bone for Gene Expression Analysis

用于基因表达分析的小鼠胚胎软骨和骨骼的激光捕获微分

Laser capture microdissection (LCM) is a powerful tool to isolate specific cell types or regions of interest from heterogeneous tissues. The cellular and ...

Transillumination-Assisted Dissection of Specific Stages of the Mouse Seminiferous Epithelial Cycle for Downstream Immunostaining Analyses

下游免疫污染分析小鼠水母上皮周期特定阶段的转化-辅助解剖

Spermatogenesis is a unique differentiation process that ultimately gives rise to one of the most distinct cell types of the body, the sperm. ...

Development of Organoids from Mouse Pituitary as In Vitro Model to Explore Pituitary Stem Cell Biology

Development of Organoids from Mouse Pituitary as In Vitro Model to Explore Pituitary Stem Cell Biology

从小鼠垂体中开发类器官作为 体外 模型以探索垂体干细胞生物学

The pituitary is the master endocrine gland regulating key physiological processes, including body growth, metabolism, sexual maturation, reproduction, ...