感谢 Rosanna Calderon、Dominic Shayler 和 Rosa Sierra 的技术建议。这项研究在一定程度上得到了南加州大学凯克医学院眼科的无限制赠款，包括预防失明研究 （AN）、美国国立卫生研究院 （NIH） K08EY030924、拉斯马德里纳斯眼科实验治疗学基金会 （AN）、预防失明研究职业发展奖 （AN）、骑士圣殿骑士眼科基金会基金会捐赠基金 （AN）、 以及 Edward N. 和 Della L. Thome 纪念基金会 （AN， KG）。

所有程序均按照南加州大学机构动物护理和使用委员会 （IACUC） 的规定执行并得到其批准。14 （n = 14） 4 至 6 个月大的荷兰带兔用于该协议的开发。雄性和雌性动物均被使用。所有动物的体重在 2.0 到 2.5 公斤之间。所有的动物都是单独饲养的。推荐的材料和设备清单可在 材料表中找到。
1. 准备工作
<ol><li>固定剂制备。<ol><li>在磷酸盐缓冲盐水 （PBS） 中制备 4% 多聚甲醛 （PFA）。每只眼睛至少需要 50 mL 固定剂。</li>
	</ol></li>
	<li>冷冻保护剂的制备。<ol><li>在PBS中制备30%体积重量（w / v）蔗糖。放在摇荡器上至少 15 分钟，以确保蔗糖完全溶解。每只眼睛至少需要 50 mL 的冷冻保护剂。</li>
	</ol></li>
	<li>解剖器械的准备。<ol><li>收集两把镊子、一把弯曲的剪刀、一把手术刀刀片和一个 100 毫米的解剖皿，并将它们放在解剖显微镜附近。设置示例如图 1A 所示。</li>
	</ol></li>
</ol>2. 初始固定
<ol><li>经结膜摘除术11 后，立即将眼睛置于冰上至少 50 mL 的 4% PFA PBS 溶液中。确保眼睛完全浸没。如果气泡聚集在眼睛的外表面，请轻轻摇晃将其清除。</li>
</ol>3. 角膜和虹膜切除
<ol><li>15分钟后，从PBS中的4%PFA中取出眼睛，并将其置于解剖显微镜下的100mm解剖皿中。用 PBS 填充培养皿，以防止眼睛干燥。</li>
	<li>切除角膜。<ol><li>首先在角膜缘前方 1 毫米处用手术刀片切开一个切口，平行于虹膜平面，以避免意外刺穿下层结构（图 1B）。</li>
		<li>将弯曲的剪刀插入初始切口，并继续沿圆周切割，同时距离角膜缘保持 1 毫米。切割时使用镊子稳定眼睛（图1C）。</li>
		<li>圆周切口完成后，用镊子取出角膜，并用实验室擦拭物轻轻擦去多余的 PBS。将角膜置于室温 （RT） 的 30% 蔗糖溶液中进行冷冻保护或丢弃。当角膜下沉时，冷冻保护就完成了，这通常需要不到 1 小时。</li>
	</ol></li>
	<li>取下虹膜。<ol><li>首先用弯曲的剪刀在虹膜上进行初步剪切。通过类似于角膜切除的圆周切割，将虹膜与组织的其余部分完全分离（图1D）。</li>
		<li>圆周切割完成后，用镊子取出虹膜，并用实验室擦拭物轻轻擦去多余的 PBS。将鸢尾花置于室温下的 30% 蔗糖溶液中进行冷冻保护或如上所述丢弃。当虹膜下沉时，冷冻保护就完成了，这通常需要不到 30 分钟的时间。</li>
	</ol></li>
</ol>4. 完成固定
<ol><li>去除角膜和虹膜后，将眼睛放入至少 50 mL 新鲜制备的 4% PFA PBS 溶液中，然后将其放在摇摇杯上轻轻搅拌。确保眼睛在搅拌过程中保持浸没。</li>
	<li>将眼睛保持在4%PFA的PBS溶液中，在4°C过夜。 注：我们发现，如果在4°C下使用16小时至24小时，4%PFA可产生出色的IHC染色，较长的固定持续时间导致非特异性背景染色和表位掩蔽增加。</li>
</ol>5. 镜片摘除
<ol><li>第二天早上，从PBS中的4%PFA中取出眼睛，并将其置于解剖显微镜下的100mm解剖皿中。用 PBS 填充培养皿，以防止眼睛干燥。</li>
	<li>取下镜头。<ol><li>首先用镊子小心地抓住前晶状体囊。 注意： 要格外小心，不要用镊子强行推拉镜片，因为这会导致视网膜脱离。</li>
		<li>小心地将剪刀插入后房，刀片沿镜片向后弯曲。用弯曲的剪刀初步切开镜片区域。 注意：要非常小心，不要用剪刀强行推动或拉动带，因为这会导致视网膜脱离（图1E）。</li>
		<li>继续以圆周图案在晶状体分区上进行小切口。每小时进行三到五次切割。</li>
		<li>要检查晶状体是否与组织的其余部分分离，请用镊子轻轻抓住晶状体前囊，并尝试轻轻提起。如果晶状体没有立即抬起，请勿尝试拉更多，因为这会导致视网膜脱离。取而代之的是，在剩余的镜头区域进行更多切割并重新尝试。</li>
		<li>一旦与其他组织分离，将镜片在室温下置于 30% 蔗糖溶液中进行冷冻保护或如上所述丢弃。当镜头下沉时，冷冻保护就完成了，这通常需要几个小时。</li>
	</ol></li>
	<li>将眼睛置于至少50mL的30%蔗糖PBS溶液中，温度为4°C，以提供冷冻保护。当眼睛下沉时，冷冻保护就完成了，这通常需要 24-48 小时。为了加快冷冻保护，在 8-12 小时后将初始蔗糖溶液更换为新鲜溶液和/或在室温下将眼睛置于蔗糖溶液中。</li>
</ol>6. 嵌入
<ol><li>冷冻保护完成后，将眼睛从 30% 蔗糖溶液中取出，并将其置于解剖显微镜下的 100 mm 解剖皿中。小心地翻转眼睛，使其朝下，以去除眼睛内部多余的蔗糖溶液。 注意：请勿尝试使用实验室湿巾从眼睛内部去除多余的溶液，因为湿巾会粘在玻璃体上并导致视网膜脱离。</li>
	<li>眼睛朝下，用湿巾轻轻擦去眼睛外部多余的蔗糖溶液。</li>
	<li>用最佳切割温度 （OCT） 化合物填充一次性包埋模具，深度为 0.5-1.0 厘米。 注意：此底座可确保眼睛不会接触模具底部，这会干扰以后的冷冻切片。</li>
	<li>将眼睛面朝上放入一次性包埋模具中。用 OCT 慢慢填充眼睛内部，特别注意避免眼睛内形成气泡。如果确实形成气泡，请小心地将其清除或使用镊子将它们推到模具的边缘。</li>
	<li>用 OCT 完成填充一次性包埋模具。 确保眼睛完全浸没在 OCT 中，并且没有接触模具的内表面或暴露在空气中。将含有眼睛的一次性包埋模具包裹在OCT中，用石蜡膜包裹;在4°C下放置过夜。 注意：根据我们的经验，较短的潜伏期不允许 OCT 完全浸润玻璃体。因此，一层蔗糖通常保留在视网膜和 OCT 之间。当这种情况发生时，在冷冻切片过程中，视网膜通常会从其余组织中分离或完全撕裂，因为冷冻的蔗糖可能比冷冻的 OCT 提供更少的结构支持，使高质量的切片难以甚至无法获得。</li>
	<li>第二天早上，将包含眼睛的包埋模具在OCT中放入100mm解剖皿中，置于解剖显微镜下。确认后透明质膜似乎从内视网膜向上抬起。 注意：这种向上的提升可能会遮挡视网膜的视野，使方向难以确定。</li>
	<li>为了更好地确定眼睛的方向，在OCT期间，请小心地向前抓住后叶状膜，并尝试用镊子将其剥离，以露出下面的视网膜。 注意：有时，透明管可能是可见的，它附着在视神经头上，并且由于视神经头位于视网膜的上部，因此可用于定向目的（图2A）。没有必要完全去除后透明膜，因为OCT已经渗入玻璃体以衬托内视网膜，任何剩余的膜都不会影响冷冻切片（图2B）。如有必要，仅应移除后透明膜以进行定向。确定方向的其他选择包括在摘除术前放置缝合线或其他类型的标记。</li>
	<li>将眼睛转移到新的包埋模具上，将新的 OCT 填充到 0.5-1.0 cm 的深度，然后按照步骤 6.3-6.5 填充新的 OCT，以确保最终包埋介质没有碎屑和/或其他可能在一夜之间积累的缺陷。如果可能，也用湿巾从眼睛内部轻轻去除 OCT，尽管这不是必需的。</li>
	<li>为模具贴上标签以进行定向（图2C）。在冷冻模具中将眼睛朝上定向可确保 OCT 与内视网膜对齐。附着在视神经头上的透明体管是确定眼睛上部的重要标志（图2A）。</li>
	<li>将包含眼睛的OCT中的标记包埋模具放在干冰上。确保眼睛没有接触嵌入模具的任何内表面或暴露在空气中。确保模具直接接触干冰，以加快冷冻过程（图2D）。</li>
	<li>将嵌入的眼睛转移到-80°C冰箱中或直接转移到低温恒温器进行切片（图2E）。</li>
</ol>7. 冷冻切片
<ol><li>安装块时，眼睛的上部朝向上方，眼睛的下部朝向下方，其余角膜和虹膜朝向右侧（ 如图 2E 所示）或左侧（未显示）。或者，根据需要以不同的方向安装块。如有必要，通过小心地一次折断模具的每一侧并直接标记块来打破模具以动员块，以免失去眼睛的方向。</li>
	<li>让块与低温恒温器内的温度平衡 30-60 分钟;-20 °C 是一个很好的起点。</li>
	<li>安装后，将新刀片放置在所需角度，通常平行于眼睛的垂直轴。</li>
	<li>根据需要设置切片厚度：10-20 μm 厚的切片是一个很好的起点。图 3A-C 演示了 14 μm 厚切片的示例。<ol><li>以缓慢且可控的方式，开始对块进行切片。确保刀片平行于眼睛的垂直轴切割块。如果没有，请相应地调整刀片与块或载物台方向接触的角度。</li>
		<li>在对块进行切片时，用刷子防止该块起皱或卷曲。快速将该部分翻转过来，然后用长毛刷轻轻向下按压，使该部分变平，并最大限度地减少皱纹或卷曲。制作一个带有尖头的单独刷子，以帮助操作该部分。</li>
	</ol></li>
	<li>通过将载玻片面朝下缓慢地朝向该部分，将该部分连接到带电的载玻片上。通过在该部分上轻轻滚动载玻片，防止该部分在安装过程中自行折叠或以其他方式变形。 注意： 请勿将滑轨直接悬停在该部分上方或将其压在该部分上，因为这可能会损坏或扭曲该部分。</li>
	<li>让载玻片干燥过夜，然后转移到-80°C冰箱或直接进行标准免疫荧光实验方案。</li>
</ol>

用于免疫组织化学研究的兔眼解剖和冷冻切片

<ol>
	<li>Peiffer, R. L., Pohm-Thorsen, L., Corcoran, K., Manning, P. J., Ringler, D. H., Newcomer, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+19-Models+in+ophthalmology+and+vision+research.">Chapter 19-Models in ophthalmology and vision research.</a> American College of Laboratory Animal Medicine, The Biology of the Laboratory Rabbit. , 409-433 (1994).</li><li>Davis, F. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+anatomy+and+histology+of+the+eye+and+orbit+of+the+rabbit.">The anatomy and histology of the eye and orbit of the rabbit.</a> Trans Am Ophthalmol Soc. 27, 400.2-441 (1929).</li><li>Zernii, E. 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=Rabbit+models+of+ocular+diseases:+New+relevance+for+classical+approaches.">Rabbit models of ocular diseases: New relevance for classical approaches.</a> CNS &amp; Neurological Disorders Drug Targets. 15 (3), 267-291 (2016).</li><li>Coons, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Labelled+antigens+and+antibodies.">Labelled antigens and antibodies.</a> Annu Rev Microbiol. 8, 333-352 (1954).</li><li>Coons, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+antibodies+as+histochemical+tools.">Fluorescent antibodies as histochemical tools.</a> Fed Proc. 10 (2), 558-559 (1951).</li><li>Coons, A. H., Kaplan, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localization+of+antigen+in+tissue+cells;+improvements+in+a+method+for+the+detection+of+antigen+by+means+of+fluorescent+antibody.">Localization of antigen in tissue cells; improvements in a method for the detection of antigen by means of fluorescent antibody.</a> J Exp Med. 91 (1), 1-13 (1950).</li><li>Pang, 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=Step-by-step+preparation+of+mouse+eye+sections+for+routine+histology,+immunofluorescence,+and+RNA+in+situ+hybridization+multiplexing.">Step-by-step preparation of mouse eye sections for routine histology, immunofluorescence, and RNA in situ hybridization multiplexing.</a> STAR Protoc. 2 (4), 100879 (2021).</li><li>Sorden, S. 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=Spontaneous+background+and+procedure-related+microscopic+findings+and+common+artifacts+in+ocular+tissues+of+laboratory+animals+in+ocular+studies.">Spontaneous background and procedure-related microscopic findings and common artifacts in ocular tissues of laboratory animals in ocular studies.</a> Toxicol Pathol. 49 (3), 569-580 (2021).</li><li>Margo, C. E., Lee, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fixation+of+whole+eyes:+the+role+of+fixative+osmolarity+in+the+production+of+tissue+artifact.">Fixation of whole eyes: the role of fixative osmolarity in the production of tissue artifact.</a> Graefes Arch Clin Exp Ophthalmol. 233 (6), 366-370 (1995).</li><li>Chatterjee, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Artefacts+in+histopathology.">Artefacts in histopathology.</a> J Oral Maxillofac Pathol. 18 (Suppl 1), S111-S116 (2014).</li><li>Mitchell, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enucleation+in+companion+animals.">Enucleation in companion animals.</a> Ir Vet J. 61 (2), 108-114 (2008).</li><li>Kastan, N. R., 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=Development+of+an+improved+inhibitor+of+Lats+kinases+to+promote+regeneration+of+mammalian+organs.">Development of an improved inhibitor of Lats kinases to promote regeneration of mammalian organs.</a> Proc Natl Acad Sci U S A. 119 (28), e2206113119 (2022).</li><li>Baiza-Dur&#225;n, 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=Safety+and+tolerability+evaluation+after+repeated+intravitreal+injections+of+a+humanized+anti-VEGF-A+monoclonal+antibody+(PRO-169)+versus+ranibizumab+in+New+Zealand+white+rabbits.">Safety and tolerability evaluation after repeated intravitreal injections of a humanized anti-VEGF-A monoclonal antibody (PRO-169) versus ranibizumab in New Zealand white rabbits.</a> Int J Retina Vitreous. 6, 32 (2020).</li><li>Yu, D. Y., Cringle, S. J., Su, E., Yu, P. K., Humayun, M. S., Dorin, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser-induced+changes+in+intraretinal+oxygen+distribution+in+pigmented+rabbits.">Laser-induced changes in intraretinal oxygen distribution in pigmented rabbits.</a> Invest Ophthalmol Vis Sci. 46 (3), 988-999 (2005).</li><li>Faude, F., 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=Facilitation+of+artificial+retinal+detachment+for+macular+translocation+surgery+tested+in+rabbit.">Facilitation of artificial retinal detachment for macular translocation surgery tested in rabbit.</a> Invest Ophthalmol Vis Sci. 42 (6), 1328-1337 (2001).</li></ol>

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在实施上述协议之前，我们在用于 IHC 的兔眼组织处理方面一直面临困难。我们已经从小鼠等较小动物的眼睛中采用了几种方案，但发现这些方案会导致固定不足和组织切片困难。有几个重要的考虑因素可以使兔子视网膜的切片保持一致、高质量。
一个考虑因素是与其他常用的动物模型（如啮齿动物）相比，兔子球体的尺寸较大。由于兔眼的大小与人眼相似，因此可以很容易...

视网膜由眼睛内的几层特化细胞组成，这些细胞共同将光转化为神经信号。由于视网膜在视力中起着至关重要的作用，因此了解其结构和功能可以为视力丧失的一些最常见原因提供有价值的见解，例如黄斑变性和糖尿病性视网膜病变等。
兔子在视网膜研究中是一种方便的动物模型，因为与其他模型相比，它们具有多种优势。兔子的眼睛在解剖结构上与人类的眼睛相对相似 1,2。例如，兔子有一个光感受器密度增加的区域，称为水平视觉条纹，类似于人类的中央凹。其他常用的动物模型，如啮齿动物，没有解剖学上的等效物。此外，与啮齿动物相比，兔子的视网膜脉管系统与人类的视网膜血管系统相当相似。兔子的眼睛也比较大。这使得它们特别适用于涉及玻璃体或视网膜内的药物给药或手术干预的研究，否则在较小的眼睛中可能难以或不可能3.
免疫组织化学 （IHC） 是一种广泛使用的技术，用于研究组织内抗原的定位，在视网膜研究中具有广泛的应用 4,5,6。由于视网膜是一个微妙的结构，通过 IHC 获得有用的结果需要仔细的组织处理。视网膜脱离和其他组织伪影（如视网膜破裂或褶皱）通常发生在处理过程中，可能会干扰结果的解释。成功的处理取决于多种因素，包括组织操作、固定的类型和持续时间、包埋介质的类型和切片技术 7,8,9,10。尽管在视网膜研究中使用兔子作为动物模型具有优势，但很少有描述兔子视网膜成功组织处理的协议。本文描述了一种从整个兔眼获得高质量视网膜切片以用于 IHC 的可靠方法。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>100 mm culture dish</td>
			<td>Corning</td>
			<td>353025</td>
			<td>Used for dissection (steps 1.3, 3, and 5)</td>
		</tr>
		<tr>
			<td>50 mL tube</td>
			<td>Genesee Scientific</td>
			<td>28-106</td>
			<td>For fixation and cryoprotection (step 1)</td>
		</tr>
		<tr>
			<td>Cryostat</td>
			<td>Leica</td>
			<td>CM1850</td>
			<td>For cryosectioning (step 7)</td>
		</tr>
		<tr>
			<td>Curved scissors</td>
			<td>Fine Science Tools</td>
			<td>91500-09</td>
			<td>Used for dissection (steps 1.3, 3, and 5)</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Fisher Scientific</td>
			<td>D3571</td>
			<td>Diluted 1:1,000 in blocking buffer</td>
		</tr>
		<tr>
			<td>Dissection microscope</td>
			<td>Zeiss</td>
			<td>Stemi 2000-C</td>
			<td>Used for dissection (steps 1.3, 3, and 5)</td>
		</tr>
		<tr>
			<td>Donkey anti-Goat 488</td>
			<td>Fisher Scientific</td>
			<td>A-11055</td>
			<td>Diluted 1:1,000 in blocking buffer</td>
		</tr>
		<tr>
			<td>Donkey anti-Mouse 555</td>
			<td>Fisher Scientific</td>
			<td>A-31570</td>
			<td>Diluted 1:1,000 in blocking buffer</td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Fine Science Tools</td>
			<td>91150-20</td>
			<td>Used for dissection (steps 1.3, 3, and 5)</td>
		</tr>
		<tr>
			<td>Glass Slide Cover</td>
			<td>VWR</td>
			<td>48404-453</td>
			<td>For cryosectioning (step 7)</td>
		</tr>
		<tr>
			<td>Goat anti-SOX2</td>
			<td>R&#38;D Systems</td>
			<td>AF2018</td>
			<td>Diluted 1:100 in blocking buffer</td>
		</tr>
		<tr>
			<td>High-profile disposable cryostat blades</td>
			<td>Leica Microsystems Inc.</td>
			<td>14035838926</td>
			<td>For cryosectioning (step 7)</td>
		</tr>
		<tr>
			<td>Kimwipe</td>
			<td>Fisher Scientific</td>
			<td>06-666-A</td>
			<td>Used to wipe away excess PBS or OCT (steps 3 and 6)</td>
		</tr>
		<tr>
			<td>Mouse anti-RPE65</td>
			<td>Novus Bio</td>
			<td>NB100-355SS</td>
			<td>Diluted 1:100 in blocking buffer</td>
		</tr>
		<tr>
			<td>OmniPur Sucrose</td>
			<td>Millipore</td>
			<td>167117</td>
			<td>Used for cryoprotectant (step 1.2)</td>
		</tr>
		<tr>
			<td>Paraformaldehyde 20% solution</td>
			<td>Electron Microscopy Sciences</td>
			<td>15713</td>
			<td>Used as tissue fixative (diluted to 4% in step 1.1)</td>
		</tr>
		<tr>
			<td>Peel-A-Away Disposable Embedding Mold (22x22x20 mm Deep)</td>
			<td>Polysciences, Inc.</td>
			<td>18646A</td>
			<td>Used as embedding mold (step 6)</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline, 1x</td>
			<td>Corning</td>
			<td>21-030-CV</td>
			<td>Used in preparation of fixative (step 1.1) and cryoprotectant (step 1.2)</td>
		</tr>
		<tr>
			<td>Scalpel blade no. 15</td>
			<td>Feather</td>
			<td>08-916-5D</td>
			<td>Used for dissection (steps 1.3, 3, and 5)</td>
		</tr>
		<tr>
			<td>Superfrost Plus Microscope Slides</td>
			<td>Fisher Scientific</td>
			<td>12-550-15</td>
			<td>For cryosectioning (step 7)</td>
		</tr>
		<tr>
			<td>Tissue-Tek O.C.T. Compound</td>
			<td>Sakura</td>
			<td>4583</td>
			<td>Used as embedding media (step 6)</td>
		</tr>
	</tbody>
</table>

obtaining high-quality cryosections of whole rabbit eye 

该协议描述了如何在较大的动物（如兔子）中获得高质量的视网膜冷冻切片。摘除后，将眼睛短暂浸入固定剂中。然后，去除角膜和虹膜，将眼睛放置过夜，以便在4°C下进行额外固定。 固定后，将镜片取下。然后将眼睛放入冷冻模型中，并填充嵌入培养基。通过去除晶状体，嵌入介质可以更好地进入玻璃体，并导致更好的视网膜稳定性。重要的是，眼睛应在包埋培养基中孵育过夜，以使眼睛完全浸润整个玻璃体。孵育过夜后，将眼睛冷冻在干冰上并切片。可以获取整个视网膜切片用于免疫组化。标准染色方案可用于研究视网膜组织内抗原的定位。遵守该协议可产生高质量的视网膜冷冻切片，可用于任何利用免疫组织化学的实验。

The retina is composed of several layers of specialized cells within the eye that together work to convert light into neural signals. Because the retina plays a critical role in vision, understanding its structure and function can provide valuable insights into some of the most common causes of vision loss such as macular degeneration and diabetic retinopathy, among others.
Rabbits serve as a convenient animal model in retinal research as they offer several advantages compared to other models. Rabbit eyes are relatively similar in anatomy to human eyes1,2. For example, rabbits have an area of increased photoreceptor density, known as the horizontal visual streak, that is analogous to the fovea in humans. Other commonly used animal models, such as rodents, do not have an anatomic equivalent. In addition, compared to rodents, the retinal vasculature in rabbits is fairly similar to that in humans. Rabbit eyes are relatively large as well. This makes them particularly suitable for studies that involve drug administration or surgical intervention within the vitreous or retina that may otherwise be difficult or impossible in a smaller eye3.
Immunohistochemistry (IHC) is a widely used technique to study the localization of antigens within a tissue and has broad applications in retinal research4,5,6. Because the retina is a delicate structure, obtaining useful results via IHC requires careful tissue processing. Retinal detachment and other tissue artifacts such as retinal breaks or folds commonly occur during processing and may interfere with the interpretation of results. Successful processing depends on a variety of factors, including tissue manipulation, type and duration of fixation, type of embedding media, and sectioning techniques7,8,9,10. Despite the advantages of using rabbits as an animal model in retinal research, very few protocols describing successful tissue processing of the rabbit retina exist. This paper describes a reliable method for obtaining high-quality retinal sections from whole rabbit eyes for use in IHC.

作者聚焦：优化兔模型中免疫组织化学的视网膜组织处理

获得全兔眼的高质量冷冻切片 

Our research focuses on novel approaches to regenerating the retinal pigment epithelium. Dysfunction of the retinal pigment epithelium is thought to play a central role in the pathophysiology of many eye diseases, such as macular degeneration, so regenerating this layer of cells is attractive from a therapeutic standpoint. Visualizing the structural anatomy of the retina via immunohistochemistry has wide applications in retinal research.In our laboratory, we found obtaining useful results from rabbit eyes, difficult due to tissue artifacts like retinal attachments and retinal folds. Despite these challenges, there are very few protocols existing that describe successful tissue processing of rabbit retina. This protocol makes several important modifications to standard tissue processing techniques that result in excellent tissue sections of the rabbit retina.This study may allow researchers to obtain better results from their research that involves the use of immunohistochemistry in rabbit models.

在组织处理后，可以使用标准的免疫荧光方案来研究视网膜内任意数量的生物过程。图3A-C显示了通过共聚焦显微镜获得的视网膜切片的代表性荧光图像。视网膜切片根据先前描述的方案12 进行免疫染色。
图3A-C所示的代表性视网膜切片位于视神经头附近，并与视?...

Read this Scientific Article Protocol about Author Spotlight: Optimizing Retinal Tissue Processing for Immunohistochemistry in Rabbit Models at JoVE.com

该协议描述了一种获得整个兔眼的高质量冷冻切片的可靠方法。它详细介绍了兔眼的解剖、固定、嵌入和切片程序，这些程序可以很容易地用于任何利用免疫组化在大眼睛中的研究。

This protocol describes how to obtain high-quality retinal cryosections in larger animals, such as rabbits. After enucleation, the eye is briefly immersed ...

我们的研究重点是再生视网膜色素上皮的新方法。视网膜色素上皮功能障碍被认为在许多眼部疾病（如黄斑变性）的病理生理学中起着核心作用，因此从治疗的角度来看，这层细胞的再生很有吸引力。通过免疫组织化学可视化视网膜的结构解剖结构在视网膜研究中具有广泛的应用。在我们的实验室中，我们发现从兔子的眼睛中获得有用的结果，由于视网膜附着和视网膜褶皱等组织伪影而很困难。尽管存在这些挑战，但很少有协议描述兔视网膜的成功组织处理。该协议对标准组织处理技术进行了几项重要的修改，从而产生了兔视网膜的出色组织切片。这项研究可能使研究人员从他们的研究中获得更好的结果，这些研究涉及在兔子模型中使用免疫组织化学。

obtaining-high-quality-cryosections-of-whole-rabbit-eye

Research

JoVE Journal

Biology

Obtaining High-Quality Cryosections of Whole Rabbit Eye 

1.1K 次观看.  University of Southern California. 我们的研究重点是再生视网膜色素上皮的新方法。视网膜色素上皮功能障碍被认为在许多眼部疾病（如黄斑变性）的病理生理学中起着核心作用，因此从治疗的角度来看，这层细胞的再生很有吸引力。通过免疫组织化学可视化视网膜的结构解剖结构在视网膜研究中具有广泛的应用。在我们的实验室中，我们发现从兔子的眼睛中获得有用的结果，由于视网膜附着和视网膜?...

视频: 作者聚焦：优化兔模型中免疫组织化学的视网膜组织处理

This protocol describes how to obtain high-quality retinal cryosections in larger animals, such as rabbits. After enucleation, the eye is briefly immersed in the fixative. Then, the cornea and iris are removed and the eye is left overnight for additional fixation at 4 &#176;C. Following fixation, the lens is removed. The eye is then placed in a cryomold and filled with an embedding medium. By removing the lens, the embedding medium has better access to the vitreous and leads to better retinal stability. Importantly, the eye should be incubated in embedding medium overnight to allow complete infiltration throughout the vitreous. Following overnight incubation, the eye is frozen on dry ice and sectioned. Whole retinal sections may be obtained for use in immunohistochemistry. Standard staining protocols may be utilized to study the localization of antigens within the retinal tissue. Adherence to this protocol results in high-quality retinal cryosections that may be used in any experiment utilizing immunohistochemistry.

This protocol describes a reliable method for obtaining high-quality cryosections of whole rabbit eyes. It details rabbit eye dissection, fixation, embedding, and sectioning procedures, which may be easily adapted for use in any study utilizing immunohistochemistry in larger eyes.

科研

生物学

Whole Rabbit Eye Dissection and Ocular Tissue Preparation for Histological Analysis

After transconjunctival enucleation of the rabbit eye, immediately place in 50 milliliters of 4%paraformaldehyde and PBS on ice. After 15 minutes, transfer the eye to a 100 millimeter dissection dish and fill the dish with PBS to prevent the eye from drying out. Under a dissection microscope, Using a surgical blade, start making an incision one millimeter anterior to the limbus, keeping it parallel to the iris plane.Insert curved scissors into the initial incision and continue cutting circumferentially, maintaining a one millimeter distance from the limbus. Then remove the cornea with forceps and gently wipe away PBS With a laboratory wipe. Place the cornea in 30%sucrose solution at room temperature for cryoprotection.Make an initial cut with curved scissors through the iris. Then cut circumferentially around the eye, similar to the cornea removal, to separate the iris from the rest of the tissue. Remove the iris with forceps and gently wipe away excess PBS with the laboratory wipe.Place the iris in 30%sucrose solution at room temperature for cryoprotection. After removing the cornea and iris, place the eye in 50 milliliters of 4%paraformaldehyde and put it on a shaker for gentle agitation. Leave the eye in 4%paraformaldehyde at four degrees Celsius overnight.The next day, place the eye in a dissection dish and fill the dish with PBS. Under the dissection microscope, use forceps to carefully grasp the anterior lens capsule. Then carefully insert the scissors into the posterior chamber, orienting the blades posteriorly along the lens.Make an initial cut through the lens zonules with curved scissors. Continue making small cuts in a circumferential pattern through the lens zonules. To confirm lens removal, gently grasp the anterior lens capsule with forceps and attempt to lift it.Upon separation, place the lens in 30%sucrose solution at room temperature for cryoprotection. Place the eye in 50 milliliters of 30%sucrose and PBS at four degrees Celsius for 24 to 48 hours for cryoprotection. To expedite cryoprotection, replace the initial sucrose solution with a fresh one after eight to 12 hours.

用于组织学分析的全兔眼解剖和眼组织制备

Ocular Tissue Embedding and Cryosectioning in Optimal Cutting Temperature Compound for Fluorescence Imaging 

After cryo protection of the rabbit eye is complete, transfer it from a 30%sucrose solution to a 100 millimeter dissection dish. Under a dissecting microscope, carefully flip the eye to remove excess sucrose solution from the inner part of the eye. Using a wipe, gently wipe away excess sucrose solution from the outer part of the eye, fill a disposable embedding mold with optimal cutting temperature or OCT compound to a depth of 0.5 to one centimeter.Place the eye in the embedding mold facing upward and slowly fill the inner part of the eye with OCT compound, avoiding bubble formation. Once the eye is emerged in the OCT compound, and does not touch the inner surfaces of the mold, wrap the embedding mold in paraffin and store it at four degrees Celsius overnight. The next day, place the embedding mold in a dissection dish.If possible, gently remove the OCT compound from the inside of the eye with a wipe. Transfer the eye to a new embedding mold filled with fresh OCT compound. Then fill the mold as previously done to ensure the final embedding medium is free of debris or imperfections.Label the mold for orientation purposes. Position the eye upward in the cryo mold, so the OCT compound lines the inner retina. Use the optic nerve head as a landmark for determining the superior part of the eye Place the labeled embedding mold on dry ice, ensuring that the eye is not touching the inner surfaces of the mold or exposed to air.Mount the block on the cryostat with the superior part of the eye facing up and the inferior part down. With the remaining cornea and iris facing to the right or left. Position a fresh blade parallel to the vertical axis of the eye to section it.After cutting the sections and drying overnight, place a slide in a freezer at 80 degrees Celsius. The fluorescence images of a whole retinal cryosection of rabbit eye demonstrated a uniform pigmented retinal pigment epithelium layer without significant detachment from the overlying photoreceptor layer. A bright-field image of the retinal section further demonstrates intact retinal morphology of all layers.