Name: isolation of intact eyeball to obtain integral ocular surface tissue for histological examination and immunohistochemistry
Uploaded: 2019-10-20T13:00:00
Description: Watch this Scientific Journal Video about Isolation of Intact Eyeball to Obtain Integral Ocular Surface Tissue for Histological Examination and Immunohistochemistry at JoVE.com

作者感谢荣居教授对手稿的批判性阅读，并感谢所有实验室成员提供技术支持。这项工作得到了国家自然科学基金（国家自然科学基金委员会：31571077）的资助;中国北京），广州市科技创新项目（201707020009）;中国广东省广州市"百人计划"来自中山大学（8300-18821104）;中国广东省广州市，中山眼科中心眼科国家重点实验室（303060202400339）;中国广东省广州市）。

所有涉及使用小鼠的程序均获得中山眼科中心动物护理和使用委员会的批准，并遵守《关于在眼科和视觉研究中使用动物的ARVO声明》。
1. 眼睑表面和眼睑完整可分光的眼球
<ol><li>切除头部。<ol>
<li>通过宫颈脱位，将产后第10天（P10）和P28小鼠（见小鼠菌株材料表）用一把锋利的剪刀砍下颈部。</li>
		<li>沿着下垂中线沿下垂中线将头分叉到鼻腔（?...

<ol>
	<li>Swamynathan, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ocular+surface+development+and+gene+expression.">Ocular surface development and gene expression.</a> Journal of Ophthalmology. 2013, (2013).</li><li>Arita, R., Fukuoka, S., Morishige, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+Morphology+of+the+Lipid+Layer+of+the+Tear+Film.">Functional Morphology of the Lipid Layer of the Tear Film.</a> Cornea. 36 Suppl 1, S60-S66 (2017).</li><li>Guo, D., 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=Ocular+surface+pathogenesis+associated+with+precocious+eyelid+opening+and+necrotic+autologous+tissue+in+mouse+with+disruption+of+Prickle+1+gene.">Ocular surface pathogenesis associated with precocious eyelid opening and necrotic autologous tissue in mouse with disruption of Prickle 1 gene.</a> Experimental Eye Research. 180, 208-225 (2018).</li><li>Knop, E., Knop, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anatomy+and+immunology+of+the+ocular+surface.">Anatomy and immunology of the ocular surface.</a> Chemical Immunology and Allergy. 92, 36-49 (2007).</li><li>Mizoguchi, S., 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=Ocular+surface+alkali+injury+damages+meibomian+glands+in+mice.">Ocular surface alkali injury damages meibomian glands in mice.</a> The Ocular Surface. 15 (4), 713-722 (2017).</li><li>Gipson, I. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Goblet+cells+of+the+conjunctiva:+A+review+of+recent+findings.">Goblet cells of the conjunctiva: A review of recent findings.</a> Progress in Retinal and Eye Research. 54, 49-63 (2016).</li><li>Nowell, C. S., 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=Chronic+inflammation+imposes+aberrant+cell+fate+in+regenerating+epithelia+through+mechanotransduction.">Chronic inflammation imposes aberrant cell fate in regenerating epithelia through mechanotransduction.</a> Nature Cell Biology. 18 (2), 168-180 (2016).</li><li>Nagata, M., Ohashi, Y., Ozawa, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+histochemical+study+of+the+development+of+premaxilla+and+maxilla+during+secondary+palate+formation+in+the+mouse+embryo.">A histochemical study of the development of premaxilla and maxilla during secondary palate formation in the mouse embryo.</a> Archives of Histology and Cytology. 54 (3), 267-278 (1991).</li><li>Zhang, L., 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=A+role+for+MEK+kinase+1+in+TGF-beta/activin-induced+epithelium+movement+and+embryonic+eyelid+closure.">A role for MEK kinase 1 in TGF-beta/activin-induced epithelium movement and embryonic eyelid closure.</a> The Embo Journal. 22 (17), 4443-4454 (2003).</li><li>Sharov, A. 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=Noggin+overexpression+inhibits+eyelid+opening+by+altering+epidermal+apoptosis+and+differentiation.">Noggin overexpression inhibits eyelid opening by altering epidermal apoptosis and differentiation.</a> The Embo Journal. 22 (12), 2992-3003 (2003).</li><li>Setala, N. L., Metso, J., Jauhiainen, M., Sajantila, A., Holopainen, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dry+eye+symptoms+are+increased+in+mice+deficient+in+phospholipid+transfer+protein+(PLTP).">Dry eye symptoms are increased in mice deficient in phospholipid transfer protein (PLTP).</a> The American Journal of Pathology. 178 (5), 2058-2065 (2011).</li><li>Zhang, Y., 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=Mastermind-like+transcriptional+co-activator-mediated+Notch+signaling+is+indispensable+for+maintaining+conjunctival+epithelial+identity.">Mastermind-like transcriptional co-activator-mediated Notch signaling is indispensable for maintaining conjunctival epithelial identity.</a> Development. 140 (3), 594-605 (2013).</li><li>McCauley, H. 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=TGFbeta+signaling+inhibits+goblet+cell+differentiation+via+SPDEF+in+conjunctival+epithelium.">TGFbeta signaling inhibits goblet cell differentiation via SPDEF in conjunctival epithelium.</a> Development. 141 (23), 4628-4639 (2014).</li><li>Guo, D., 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=Ocular+surface+pathogenesis+associated+with+precocious+eyelid+opening+and+necrotic+autologous+tissue+in+mouse+with+disruption+of+Prickle+1+gene.">Ocular surface pathogenesis associated with precocious eyelid opening and necrotic autologous tissue in mouse with disruption of Prickle 1 gene.</a> Experimental Eye Research. 180, 208-225 (2019).</li></ol>

准备完整眼球的一个关键提醒是，所有轨道骨骼必须完全去除，尤其是小而靠近眼窝底部的朱加骨和乳状骨骼。任何遗留的骨头都可能使随后的骨骼学复杂化。如果一小块骨头没有被意外从解剖中完全去除，则可以使用一对尖锐的钳子从嵌入的石蜡块中挑选出来。此操作后，留下的孔应填充熔化的石蜡。
处理镜头时应格外小心。破碎的镜头通常分散在整个部分。这将极大地?...

眼部表面由连续的局部上皮片组成，包括胸膜结膜、胸膜结膜和角膜1。许多腺体结构与眼表面上皮相关，并共同产生一层撕裂膜，保护角膜表面免受干燥和环境入侵2。操作系统中断会导致角膜炎、结膜炎或两者（角膜结膜炎）。遗传因素和环境刺激物或其相互作用都促成了OS3，4的病理改变。因此，各种基因工程和物理或化学诱导的动物模型被用于研究人类操作系统的疾病过程。
鼠标操作系统的结构和功能在许多方面与人类相似。眼腺分泌的泪膜成分在小鼠和人类之间也相似。利用小鼠模型阐明人类OS疾病5、6、7的机制进行了大量研究。在许多情况下，分析操作系统的全局性而不是局部分子变化，以获得相同实验处理下的全面信息至关重要。因此，需要具有良好完整性的样品制备，以确保同时分析操作系统的每个部分。
鼠标操作系统与嵌入在眼窝/轨道中的眼球（由几个不同头骨制成的骨杯）紧密相连，并通过薄连接组织连接到它。在不破坏眼皮或胸膜结膜的情况下解剖整个眼部表面存在巨大的挑战。这些挑战来自：（i） 操作系统柔软而薄薄，并贴在物理上分离但可移动的眼睑和眼球上，因此容易受到变形和损坏;（ii）轨道骨骼和眼球之间的有限空间限制了解剖操作。对于成年小鼠来说，挑战要大得多。在胚胎小鼠中，轨道骨的骨化不完备，周围组织相对松散8。头部可以去除和分一半，然后直接进行甲醛（PFA）固定和嵌入9。相比之下，产后和成年小鼠轨道骨骼完全被厚厚的周围组织所渗透，使得固定剂的渗透效率降低。此外，轨道/头骨是硬和脆的，很容易打破时，切片他们在软嵌入化合物，如石蜡。破碎的骨头会一致撕裂附近的组织，导致劣质组织形态。
许多已发表的研究往往显示部分眼表面，这可能足以为他们的特定研究目的10，11。对文献的一次总检查发现，只有很少的研究显示整个完整的眼表面被证明没有详细的解剖协议12，13。在此协议中，我们详细介绍了一种解剖方法，以获得完整的产后眼部表面，其中轨道和头骨被有序地去除，留下未接触的眼表面以及眼球和眼睑，最大限度地减少了物理损伤。我们进一步检查了OS组织学，并使用使用该协议准备的组织部分进行免疫组织化学。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1&#215; Phosphate buffered saline (PBS)</td>
			<td>Transgen Biotech</td>
			<td>FG701-01</td>
			<td></td>
		</tr>
		<tr>
			<td>50ml centrifuge tube</td>
			<td>Corning</td>
			<td>430829</td>
			<td></td>
		</tr>
		<tr>
			<td>Adhesive microscope slides</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 Phalloidin</td>
			<td>Invitrogen/Life Technologies</td>
			<td>A12379</td>
			<td>Suggested concentration 1:500 - 1,000</td>
		</tr>
		<tr>
			<td>Alexa Fluor 568 Phalloidin</td>
			<td>Invitrogen/Life Technologies</td>
			<td>A12380</td>
			<td>Suggested concentration 1:500 - 1,000</td>
		</tr>
		<tr>
			<td>Anti-K12 antibody</td>
			<td>Abcam</td>
			<td>ab124975</td>
			<td>Suggested concentration 1:1,000</td>
		</tr>
		<tr>
			<td>Anti-K14 antibody</td>
			<td>Abcam</td>
			<td>ab7800</td>
			<td>Suggested concentration 1:800</td>
		</tr>
		<tr>
			<td>Citric acid</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cover slide</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Curved forceps</td>
			<td>World Precision Instruments</td>
			<td>14127</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting microscope.</td>
			<td>Olmpus</td>
			<td>SZ61</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethyl alcohol</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescent Microscope</td>
			<td>Zeiss</td>
			<td>AxioImager.Z2</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluoromount-G Mounting media</td>
			<td>SouthernBiotech</td>
			<td>0100-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro dissecting scissors-straight blade</td>
			<td>World Precision Instruments</td>
			<td>503242</td>
			<td></td>
		</tr>
		<tr>
			<td>Microwave ovens</td>
			<td>Galanz</td>
			<td>P70D20TL-D4</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse strains</td>
			<td>C57/BL6 and Sv129 mixed</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>No.4 straight forceps</td>
			<td>World Precision Instruments</td>
			<td>501978-6</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal Goat Serum</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde (PFA)</td>
			<td>Various</td>
			<td></td>
			<td>Prepare a 4% solution in 1&#215; PBS and filter with 0.45&#956;m filter membrane</td>
		</tr>
		<tr>
			<td>Tissue culture dish</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Trisodium citrate</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>SLBW6818</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation of intact eyeball to obtain integral ocular surface tissue for histological examination and immunohistochemistry

眼部表面 （OS） 由上皮片组成，其三个连接部分：胸膜结膜、胸膜结膜和角膜上皮。操作系统中断会导致角膜炎、结膜炎或两者（角膜结膜炎）。在具有某些基因修饰或人工操作的实验动物模型中，检查 OS 上皮表的所有部分以并行评估每个部分的相对病理遗传变化非常有用。然而，对操作系统组织整体进行解剖，没有变形或损伤一直具有挑战性，这主要是因为附在物理上分离但可移动的眼睑和眼球的操作系统柔软和薄薄。此外，硬头骨/轨道骨骼形成的深眼窝被眼球完全占据，留下有限的空间进行解剖。因此，从面部侧直接切除眼球与相关的OS组织往往会导致组织损伤，特别是胸膜和胸膜结膜。在这个协议中，我们描述了一种从分离的小鼠头部依次取出头骨和轨道骨骼的方法，将眼睑、眼表面、透镜和视网膜完全分离在一块。OS 表的完整性得到了很好的保存，可以通过组织学或免疫染色在单个部分进行检查。

从不同角度看的主要头骨骨骼如图1A-E所示，颜色表示不同的骨骼。我们使用四周大的动物来演示解剖过程。在解剖步骤 1.1.1-1.1.3 和短前缀（步骤 1.1.4）之后，图 1E中演示了具有相关面部骨骼的眼球。进一步修剪，以去除前（鼻和上半身）和后部（例如，前体）以及食性骨骼产生的组织块，主要是眼球?...

Watch this Scientific Journal Video about Isolation of Intact Eyeball to Obtain Integral Ocular Surface Tissue for Histological Examination and Immunohistochemistry at JoVE.com

Isolation of Intact Eyeball to Obtain Integral Ocular Surface Tissue for Histological Examination and Immunohistochemistry

该协议描述了一种在相对完整位置分离眼睑、眼表面、前段和后段的小鼠眼球的方法。

分离完整眼珠以获得整体眼表面组织，用于组织学检查和免疫组织化学

Ocular surface (OS) consists of an epithelial sheet with three connected parts: palpebral conjunctiva, bulbar conjunctiva and corneal epithelium. ...

我们的实验室专注于眼部发育和疾病。该协议描述了一种从解剖的小鼠头部中按顺序取出头骨和轨道骨骼的方法。将眼睑、眼表面、透镜和视网膜放在一起。该协议将有利于对眼表面教学感兴趣的眼科医生。我是郭迪安，在解剖面部骨骼时，在试图避免眼球的损害时，告诉技术可能挣扎。这种解剖物质的主要优点是，眼表面上皮板的完整性保存完好，眼表面的不同部分可以通过组织学或免疫覆盖在一个部分检查。一定要切断软组织的所有小骨头和碎屑。原因，它会导致疤痕标记组织部分，否则。对鼠标进行安乐死后，用一把直剪刀切断头部，然后沿着下垂中线一样，从腹腔骨到鼻腔。将头的两半放入干净的培养皿中，用锋利的剪刀切断下颚和舌头。释放视神经，在视光基亚姆位置之前切断它的大脑。并删除所有脑组织，包括嗅觉球，这是连接到头骨壁。现在，所有剩余的组织应有与眼球相关的所有头骨和轨道骨骼。用 PBS 清洗其余部分，以清除任何血液和头发碎屑。将组织放在含有4%甲醛的50毫升管中，并在室温下旋转，以预先固定。前缀完成后，快速洗涤PBS中的解剖头三次，以进一步消除破碎的头发和固定。将组织放入10厘米的培养皿中，沿着文本协议图之一所示平面将头骨切成三个部分。眼球和插座隐藏在头骨的中间部分，在头骨下，正面和骨骼。使用两对四号直钳剥离 ethmoid 骨骼，并完全露出正面和最大骨骼。然后，在地理上将头骨表面划分为四个方面。将弯曲钳尖水平插入每个区域，以按顺序去除最大骨骼和正面骨骼。在此之后，底层的拉克里姆和巨骨将暴露。去除眼球周围的两块骨头和相关皮下肌肉和脂肪。将眼睑和皮肤附着的眼球修剪成一个方形的小方块，以减少组织体积，并在嵌入时方便组织定向。将眼球和相关组织放回4%的甲醛中，并在4点继续通宵修复

Research

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Biology

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 ...

科研

生物学

Production of Tissue Microarrays, Immunohistochemistry Staining and Digitalization Within the Human Protein Atlas

组织芯片，​​免疫组织化学染色和数字化生产在人类蛋白质图集

The  tissue microarray (TMA) technology provides the means for high-throughput  analysis of multiple tissues and cells. The technique is used within the ...

Avidity-based Extracellular Interaction Screening (AVEXIS) for the Scalable Detection of Low-affinity Extracellular Receptor-Ligand Interactions

Avidity-based Extracellular Interaction Screening AVEXIS for the Scalable Detection of Low-affinity Extracellular Receptor-Ligand Interactions

基于亲和力，外的相互作用筛选（AVEXIS）外低亲和力受体 - 配体相互作用的可扩展检测

Extracellular protein:protein interactions between secreted or membrane-tethered proteins are critical for both initiating intercellular communication and ...

Isolation and Differentiation of Stromal Vascular Cells to Beige/Brite Cells

米色/布里特细胞的间质血管细胞的分离和分化

Brown adipocytes have the ability to uncouple the  respiratory chain in mitochondria and dissipate chemical energy as heat.  Development of UCP1-positive ...

Dissection of the Auditory Bulla in Postnatal Mice: Isolation of the Middle Ear Bones and Histological Analysis

在出生后的小鼠听泡解剖：中耳骨骼和组织学分析的分离

In most mammals, auditory ossicles in the middle ear, including the malleus, incus and stapes, are the smallest bones. In mice, a bony structure called ...

Histological-Based Stainings Using Free-Floating Tissue Sections

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 ...

Isolation and Characterization of Intact Phycobilisome in Cyanobacteria

蓝藻中完整藻胆体的分离与表征

In cyanobacteria, phycobilisome is a vital antenna protein complex that harvests light and transfers energy to photosystem I and II for photochemistry. ...

Isolation and Analysis of Traceable and Functionalized Extracellular Vesicles from the Plasma and Solid Tissues

从血浆和实体组织中分离和分析可追溯和功能化的细胞外囊泡

Circulating and tissue-resident extracellular vesicles (EVs) represent promising targets as novel theranostic biomarkers, and they emerge as important ...

Isolation, Characterization, and Therapeutic Application of Extracellular Vesicles from Cultured Human Mesenchymal Stem Cells

培养的人间充质干细胞细胞外囊泡的分离、表征和治疗应用

Extracellular vesicles (EVs) are heterogeneous membrane nanoparticles released by most cell types, and they are increasingly recognized as physiological ...

Efficient and Consistent Generation of Retinal Pigment Epithelium/Choroid Flatmounts from Human Eyes for Histological Analysis

从人眼高效、一致地生成视网膜色素上皮/脉络膜平面安装，用于组织学分析

The retinal pigment epithelium (RPE) and retina are functionally and structurally connected tissues that work together to regulate light perception and ...