Name: quantification of immunostained caspase-9 in retinal tissue
Uploaded: 2022-07-25T15:00:00
Description: Watch this Scientific Journal Video about Quantification of Immunostained Caspase-9 in Retinal Tissue at JoVE.com

这项工作得到了美国国家科学基金会研究生研究奖学金计划（NSF-GRFP）资助DGE - 1644869和美国国立卫生研究院（NIH）国家神经疾病和中风研究所（NINDS）的支持，奖励号F99NS124180 NIH NINDS多样性专业F99（CKCO），国家眼科研究所（NEI）5T32EY013933（AMP），国家神经系统疾病和中风研究所（RO1 NS081333， R03 NS099920 to CMT）和国防部陆军/空军（DURIP到CMT）。

该协议遵循视觉和眼科研究协会（ARVO）关于在眼科和视力研究中使用动物的声明。啮齿动物实验由哥伦比亚大学机构动物护理和使用委员会（IACUC）批准和监测。
1.视网膜组织的制备和冷冻切片
<ol><li>通过给予腹膜内麻醉（氯胺酮（80-100mg / kg）和甲苯噻嗪（5-10mg / kg））对动物实施安乐死，并使用蠕动泵用4%多聚甲醛（PFA）灌注接受RVO的小鼠（详见...

<ol>
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N., 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=Caspase-2+mediates+site-specific+retinal+ganglion+cell+death+after+blunt+ocular+injury.">Caspase-2 mediates site-specific retinal ganglion cell death after blunt ocular injury.</a> Investigative Ophthalmology &amp; Visual Science. 59 (11), 4453-4462 (2018).</li><li>Akpan, N., 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=Intranasal+delivery+of+caspase-9+inhibitor+reduces+caspase-6-dependent+axon/neuron+loss+and+improves+neurological+function+after+stroke.">Intranasal delivery of caspase-9 inhibitor reduces caspase-6-dependent axon/neuron loss and improves neurological function after stroke.</a> Journal of Neuroscience. 31 (24), 8894-8904 (2011).</li><li>London, A., Benhar, I., Schwartz, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+retina+as+a+window+to+the+brain-from+eye+research+to+CNS+disorders.">The retina as a window to the brain-from eye research to CNS disorders.</a> Nature Reviews Neurology. 9 (1), 44-53 (2013).</li><li>Song, P., Xu, Y., Zha, M., Zhang, Y., Rudan, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+epidemiology+of+retinal+vein+occlusion:+a+systematic+review+and+meta-analysis+of+prevalence,+incidence,+and+risk+factors.">Global epidemiology of retinal vein occlusion: a systematic review and meta-analysis of prevalence, incidence, and risk factors.</a> Journal of Global Health. 9 (1), 010427 (2019).</li><li>Akpan, N., 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=Intranasal+delivery+of+caspase-9+inhibitor+reduces+caspase-6-dependent+axon/neuron+loss+and+improves+neurological+function+after+stroke.">Intranasal delivery of caspase-9 inhibitor reduces caspase-6-dependent axon/neuron loss and improves neurological function after stroke.</a> The Journal of neuroscience. 31 (24), 8894-8904 (2011).</li><li>Gage, G. J., Kipke, D. R., Shain, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole+animal+perfusion+fixation+for+rodents.">Whole animal perfusion fixation for rodents.</a> Journal of Visualized Experiments. (65), e3564 (2012).</li><li>McStay, G. P., Salvesen, G. S., Green, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overlapping+cleavage+motif+selectivity+of+caspases:+implications+for+analysis+of+apoptotic+pathways.">Overlapping cleavage motif selectivity of caspases: implications for analysis of apoptotic pathways.</a> Cell Death &amp; Differentiation. 15 (2), 322-331 (2007).</li><li>Kuida, K., 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=Reduced+apoptosis+and+cytochrome+c-mediated+caspase+activation+in+mice+lacking+caspase+9.">Reduced apoptosis and cytochrome c-mediated caspase activation in mice lacking caspase 9.</a> Cell. 94 (3), 325-337 (1998).</li><li>Troy, C. 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半胱天冬酶是一个多成员蛋白酶家族，最好研究它们在细胞死亡和炎症中的作用;然而，最近发现了一些家庭成员的各种非死亡功能4，5。我们对半胱天冬酶功能的大部分理解来自细胞培养的工作和人类疾病的推断数据。虽然可以理解半胱天冬酶在疾病中的异常诱导、激活或失活，但从功能上确定半胱天冬酶是否驱动疾病病理一直具有挑战性。许多用于?...

半胱天冬酶是调节疾病1，2中发育细胞死亡，免疫反应和异常细胞死亡的蛋白酶家族。虽然众所周知，半胱天冬酶家族的成员在各种神经退行性疾病中被诱导，但了解哪种半胱天冬酶驱动疾病病理学更具挑战性3。此类研究需要工具来识别、表征和验证单个半胱天冬酶家族成员的功能。从机制和治疗的角度来看，解析相关的个体半胱天冬酶都很重要，因为文献中有多项研究提供了半胱天冬酶不同作用的证据4，5。因此，如果目标是针对疾病中的半胱天冬酶以获得治疗益处，那么针对相关家庭成员的特定目标至关重要。检测组织中半胱天冬酶水平的传统技术包括蛋白质印迹和酶法和荧光法3，6。然而，这些措施都不允许对半胱天冬酶水平进行细胞特异性检测，在某些情况下，裂解的半胱天冬酶通常无法通过传统的蛋白质分析措施检测到。众所周知，半胱天冬酶可以在同一组织中发挥不同的凋亡和非凋亡作用7，因此需要仔细表征细胞特异性半胱天冬酶水平，以准确了解发育和疾病途径。
这项研究显示了半胱天冬酶在神经血管缺氧缺血 - 视网膜静脉阻塞（RVO）模型中的激活和功能7，8。在视网膜等复杂组织中，有多种细胞类型可能受到 RVO 诱导的缺氧缺血的影响，包括神经胶质细胞、神经元和脉管系统7。在成年小鼠视网膜中，通过免疫组织化学（IHC）7测量，健康组织中半胱天冬酶的表达很少，但在发育过程中 情况并非如此9或视网膜疾病模型10，11。IHC是一种在生物医学研究中成熟的技术，可以验证疾病和病理靶点，通过空间定位和蛋白质量化来识别新角色。如果蛋白质印迹或荧光分析无法检测到裂解的半胱天冬酶产物，也无法通过定位检测不同半胱天冬酶的特定细胞位置或询问半胱天冬酶信号通路，则应使用IHC。
为了确定RVO中功能相关的半胱天冬酶，将IHC与经过验证的半胱天冬酶抗体和细胞标志物一起使用。先前在实验室进行的研究表明，半胱天冬酶-9在缺血性中风和半胱天冬酶-9的抑制模型中被迅速激活，具有高度特异性的抑制剂，可防止神经元功能障碍和死亡12。由于视网膜是中枢神经系统（CNS）的一部分，因此它可作为模型系统来查询和进一步研究半胱天冬酶-9在神经血管损伤中的作用13。为此，利用RVO小鼠模型研究半胱天冬酶-9的细胞特异性位置和分布及其在神经血管损伤中的意义。RVO是由血管损伤引起的工作年龄成年人失明的常见原因14。发现半胱天冬酶-9在内皮细胞中以非凋亡方式表达，但不在神经元中表达。
作为一种组织，视网膜的优点是可以可视化为平面安装，允许欣赏血管网络，或作为横截面，突出神经元视网膜层。横截面中半胱天冬酶蛋白表达的定量提供了背景，通过识别半胱天冬酶在视网膜中的定位，半胱天冬酶在视网膜神经元连接和视觉功能中具有潜在关键意义。在鉴定和验证之后，使用鉴定的半胱天冬酶的诱导细胞特异性缺失来实现目标半胱天冬酶的靶向。对于潜在的治疗查询，使用特定工具来抑制活化的半胱天冬酶来测试感兴趣的半胱天冬酶的相关性。对于半胱天冬酶-9，细胞通透性高选择性抑制剂7，15，使用Pen1-XBIR3。对于本报告，使用了具有C57BL / 6J背景的2个月大的男性C57BL / 6J菌株和他莫昔芬诱导的内皮半胱天冬酶-9敲除（iEC Casp9KO）菌株。这些动物暴露于RVO小鼠模型，C57BL / 6J用半胱天冬酶-9选择性抑制剂Pen1-XBir3处理。所描述的方法可以应用于中枢和外周系统中的其它疾病模型7，15。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>anti-Caspase-7 488</td>
			<td>Novus Biologicals</td>
			<td>NB-56529AF488</td>
			<td>use at 1:150</td>
		</tr>
		<tr>
			<td>anti-cl-Caspase-9</td>
			<td>Cell Signaling</td>
			<td>9505-S</td>
			<td>use at 1:800</td>
		</tr>
		<tr>
			<td>anti-CD31</td>
			<td>BD Pharmingen</td>
			<td>553370</td>
			<td>use at 1:50</td>
		</tr>
		<tr>
			<td>Confocal Spinning Disc Microscope</td>
			<td>Biovision</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>FIJI 2.3.0</td>
			<td>open source</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fluormount G</td>
			<td>Fisher</td>
			<td>50-187-88</td>
			<td></td>
		</tr>
		<tr>
			<td>Forcep</td>
			<td>Roboz</td>
			<td>RS-5015</td>
			<td></td>
		</tr>
		<tr>
			<td>iCasp9FL/FL X VECad-CreERT2 mice</td>
			<td>lab generated</td>
			<td></td>
			<td>see Avrutsky 2020</td>
		</tr>
		<tr>
			<td>Isolectin (594, 649)</td>
			<td>Vector</td>
			<td>DL-1207</td>
			<td>use at 1:200</td>
		</tr>
		<tr>
			<td>Ketamine Hydrochloride</td>
			<td>Henry Schein</td>
			<td>NDC: 11695-0702-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Perfusion pump&#160;</td>
			<td>Masterflex</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pen1-XBir3</td>
			<td>lab generated</td>
			<td></td>
			<td>see Avrutsky 2020</td>
		</tr>
		<tr>
			<td>Prism 9.1</td>
			<td>GraphPad</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue-Tek O.C.T.</td>
			<td>Fisher</td>
			<td>14-373-65</td>
			<td></td>
		</tr>
		<tr>
			<td>Vis-a-View 4.0</td>
			<td>Visitron Systems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Akorn</td>
			<td>NDCL 59399-110-20</td>
			<td></td>
		</tr>
	</tbody>
</table>

quantification of immunostained caspase-9 in retinal tissue

已知半胱天冬酶家族介导细胞死亡以外的许多细胞途径，包括细胞分化、轴突寻路和增殖。自从鉴定细胞死亡蛋白酶家族以来，一直在寻找工具来识别和扩展特定家族成员在发育、健康和疾病状态下的功能。然而，目前广泛使用的许多市售半胱天冬酶工具并非特定于靶向半胱天冬酶。在本报告中，我们描述了我们用于识别，验证和靶向神经系统中caspase-9的方法，该方法使用新型抑制剂和具有免疫组织化学读数的遗传方法。具体来说，我们使用视网膜神经元组织作为模型来识别和验证半胱天冬酶的存在和功能。该方法能够询问细胞类型特异性凋亡和非凋亡半胱天冬酶-9功能，并且可以应用于其他感兴趣的复杂组织和半胱天冬酶。了解半胱天冬酶的功能有助于扩展当前细胞生物学的知识，并且由于它们参与疾病，也有利于确定潜在的治疗靶点。

所述协议允许用户分析和量化视网膜组织中的半胱天冬酶-9水平。此外，它还提供了进一步鉴定、验证和特异性靶向半胱天冬酶-9和下游底物的工具。总结的步骤允许对荧光显微照片中的半胱天冬酶水平和细胞特异性进行可量化分析。所有图均显示了代表性的显微照片和未受伤和 1 天 P-RVO 视网膜横截面中总视网膜、内皮细胞和神经元中指示的半胱天冬酶水平的量化。视网膜横截面允许在用 Hoechst ?...

Watch this Scientific Journal Video about Quantification of Immunostained Caspase-9 in Retinal Tissue at JoVE.com

Quantification of Immunostained Caspase-9 in Retinal Tissue

这里介绍详细的免疫组织化学方案，用于鉴定、验证和靶向复杂组织中功能相关的半胱天冬酶。

视网膜组织中免疫染色半胱天冬酶-9的定量

The family of caspases is known to mediate many cellular pathways beyond cell death, including cell differentiation, axonal pathfinding, and ...

该协议提供了一种详细的免疫组织化学方法来鉴定，验证和靶向复杂组织中功能相关的半胱天冬酶。该技术的主要优点是能够询问细胞类型特异性凋亡和非凋亡半胱天冬酶-9功能，并且可以应用于其他感兴趣的复杂组织和半胱天冬酶。了解半胱天冬酶的功能有助于扩展细胞生物学的当前知识，并且由于它们参与疾病，也有利于确定潜在的治疗靶点。使用染色视网膜切片的共聚焦显微镜图像开始量化半胱天冬酶水平。为此，请将 405、470、555 和 640 通道的图像文件拖到斐济的控制台上。单击“图像”、“颜色”和“合并通道”选项卡，为文件指定颜色。然后通过单击图像堆栈和 Z 项目来压缩 Z 堆栈。选择投影类型作为最大强度。通过单击图像打开通道的界面，然后单击颜色通道的工具。接下来，打开亮度和对比度界面。要选择每个通道的参数，请转到通道的选项卡并选择一个通道。将光标放在背景顶部并注释像素值。然后将光标放在半胱天冬酶表达的单元格上并注释像素值。要测试参数，请转到亮度和对比度界面，然后单击选择。插入最小显示值或半胱天冬酶表达单元格的像素值。然后在单击确定之前插入最大显示值或背景像素值。从盲组织中选择随机图像，并重复选择每个通道的参数以测试所有通道的参数。设置参数后，打开图像文件，然后压缩 Z 堆栈。添加半胱天冬酶通道的亮度和对比度参数以及血管标志物通道的异凝集素。使用点工具，使用血管标志物与半胱天冬酶表达的共定位作为阳性区域的读数来量化阳性血管区域的数量。然后使用钩子作为阳性神经元区域的标记，量化正血管区域的数量。在电子表格上为每个图像注释值，并按部分平均值。平均截面值，这将是每只眼睛的读数。该方案确定了半胱天冬酶的细胞定位和半胱天冬酶表达的神经元视网膜层。视网膜横截面允许视网膜神经节层或RGL，核间层或INL以及用钩子染色时外核层或ONL中的视网膜核的可视化。在横截面中，视网膜血管似乎断开，并在RGL和INL之间以及INL和ONL之间分离供应丛状层。视网膜横截面还确定了高度调控的半胱天冬酶-9，即视网膜静脉阻塞后一天。半胱天冬酶-9抑制在降低内皮细胞敲除小鼠神经元半胱天冬酶-7表达方面更有效，验证了内皮半胱天冬酶-9的功能相关性。代表性分析表明，半胱天冬酶-9活性的抑制在药理学上阻断了半胱天冬酶-7在视网膜静脉阻塞后的表达。该技术的成功依赖于仔细的组织制备以及IHC和显微成像，以确保数据的一致性，有效性和可靠性。该方法可与蛋白质印迹分析相结合，用于半胱天冬酶信号传导的半定量比较。该技术提供了询问RVO中靶向活性半胱天冬酶-9的治疗方法疗效的步骤。这种方法也可以应用于其他组织，包括大脑。

Research

JoVE Journal

Neuroscience

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

小鼠视网膜神经节细胞的转染在体内电穿孔

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

科研

神经科学

Morphometric Analyses of Retinal Sections

视网膜节的形态分析

Morphometric analyses of retinal sections have been used in examining retinal diseases. For examples, neuronal cells were significantly lost in the ...

Automated Quantification of Synaptic Fluorescence in C. elegans

Automated Quantification of Synaptic Fluorescence in C. elegans

突触荧光自动定量<em&gt; C。线虫</em

Synapse strength refers to the amplitude of postsynaptic responses to presynaptic neurotransmitter release events, and has a major impact on overall ...

Dissection, Culture, and Analysis of Xenopus laevis Embryonic Retinal Tissue

Dissection, Culture, and Analysis of Xenopus laevis Embryonic Retinal Tissue

解剖，文化与分析<em&gt;非洲爪蟾</em&gt;胚胎视网膜组织

The process by which  the anterior region of the neural plate gives rise to the vertebrate retina  continues to be a major focus of both clinical and ...

Quantification of Filamentous Actin (F-actin) Puncta in Rat Cortical Neurons

Quantification of Filamentous Actin F-actin Puncta in Rat Cortical Neurons

大鼠皮层神经元丝状肌动蛋白（F-肌动蛋白）泪点的量化

Filamentous actin protein (F-actin) plays a major role in spinogenesis, synaptic plasticity, and synaptic stability. Changes in dendritic F-actin rich ...

Quantification of Endosome and Lysosome Motilities in Cultured Neurons Using Fluorescent Probes

使用荧光探针定量培养的神经元中的内体和溶酶体运动

In the brain, membrane trafficking systems play important roles in regulating neuronal functions, such as neuronal morphology, synaptic plasticity, ...

Purification of H3 and H4 Histone Proteins and the Quantification of Acetylated Histone Marks in Cells and Brain Tissue

h3 和 h4 组蛋白的纯化及细胞和脑组织中乙酰化组蛋白标记的定量

In all eukaryotic organisms, chromatin, the physiological template of all genetic information, is essential for heredity. Chromatin is subject to an array ...

Preparation of Mouse Retinal Cryo-sections for Immunohistochemistry

小鼠视网膜冷冻节的制备,用于免疫性植物化学

Preparation of high-quality mouse eye sections for immunohistochemistry (IHC) is critical for assessing the retinal structure and function and for ...

Subretinal Transplantation of Human Embryonic Stem Cell-Derived Retinal Tissue in a Feline Large Animal Model

猫大型动物模型中人类胚胎干细胞来源的视网膜组织的视网膜下移植

Retinal degenerative (RD) conditions associated with photoreceptor loss such as age-related macular degeneration (AMD), retinitis pigmentosa (RP) and ...

A Hydrophobic Tissue Clearing Method for Rat Brain Tissue

鼠脑组织疏水组织清除方法

Hydrophobic tissue clearing methods are easily adjustable, fast, and low-cost procedures that allows for the study of a molecule of interest in unaltered ...