使用免疫过氧化物酶和免疫荧光方法鉴定成年斑马鱼中的奥列辛和内他大麻素受体
Summary
这里介绍的是免疫组织化学表征和定位的orexin肽,orexin受体,和内分泌大麻素受体在肠道和大脑中正常和饮食诱发肥胖(DIO)成年斑马鱼模型使用免疫渗透酶和双免疫荧光法。
Abstract
免疫组织化学(IHC)是一种高度敏感和特异性的技术,涉及在带有标记抗体的组织部分检测靶抗原。这是一个多步骤过程,其中每个步骤的优化对于获得最佳特定信号至关重要。通过IHC,可以检测出特定生物标志物的分布和定位,揭示进化保护的信息。此外,IHC还有助于了解生物标志物在病理条件下(如肥胖)的表达和分布变化。IHC,主要是免疫荧光技术,可用于成年斑马鱼检测植物遗传保护分子的组织和分布,但标准的IHC协议没有被破坏。Orexin和内分泌大麻素是两个高度保守的系统,涉及控制食物摄入量和肥胖病理学。这里报道的是用于获取有关orexin肽(OXA)、orexin受体(OX-2R)以及大麻素受体(CB1R)的定位和分布在肠道和大脑中正常和饮食诱发的肥胖(DIO)成年斑马鱼模型的信息的协议。还介绍了免疫过氧化物酶和双免疫荧光的方法,以及试剂的制备、固定、石蜡嵌入和斑马鱼组织的冷冻保护,以及内源性活动阻断步骤和背景的准备反染色。完整的参数集来自以前的IHC实验,通过这些实验,我们展示了免疫荧光如何帮助理解OX、OX-2R和CB1R在成年斑马鱼中的表达分布、定位和保存组织。生成的具有高度特异性信号强度的图像证实斑马鱼是适合于特定生物标志物分布、定位和进化保护的免疫组织化学研究的动物模型。生理和病理状况。这里介绍的协议推荐用于成人斑马鱼的IHC实验。
Introduction
免疫组织化学(IHC)是一种公认的经典技术,用于通过抗原-抗体相互作用1,2识别细胞或组织成分(抗原)。它可用于识别组织内目标生物分子的定位和分布。IHC使用免疫学和化学反应来检测组织第3部分的抗原。用于抗原-抗体相互作用可视化的主要标志物包括荧光染料(免疫荧光)和酶-基质颜色反应(免疫过氧化物酶),两者均与抗体4结合。使用微观观察可以确定标记组织的定位,这大约对应于组织中目标抗原的定位。
荧光或色原反应检测蛋白质的两种方法:直接检测方法,即直接标记特定原抗体的方法;间接检测方法,其中原抗体未结合,而二级抗体携带标签5,6,7。间接方法具有一些优点,主要是信号放大。此外,与其他分子和细胞技术不同,免疫荧光,可以可视化两个或两个以上蛋白质在细胞和组织7中不同表达的分布、定位和共表达。所使用的检测方法的选择取决于实验细节。
迄今为止,IHC被广泛用于基础研究,作为了解生物标志物的分布和定位以及从人类到无脊椎动物在生物组织中不同蛋白质的一般轮廓的有力和必要的工具。9,10,11.该技术有助于显示大量正常和改变的动物器官和不同组织类型的蛋白质表达图,显示生理和病理变化引起的表达可能向下或向上调节。IHC是一种高度敏感的技术,需要准确性和正确选择方法才能获得最佳结果12。首先,许多不同的因素,如固定,交叉反应,抗原检索,和抗体的敏感性可能导致假阳性和假阴性信号13。抗体的选择是IHC中最重要的步骤之一,取决于抗原特异性及其与被调查的蛋白质和物种的亲和力。
最近,我们优化了IHC技术,以检测成年斑马鱼组织中的奥西辛/脱脂素和内分泌大麻素系统的成员。我们主要专注于固定,组织嵌入使用两种不同的方法,切片和安装(这会影响分辨率和细节在微观分析),和阻塞(防止误报和减少背景)14。其他重要特征是单个IHC协议的抗体特异性和选择性和可重复性。提供抗体特异性的关键是使用阴性对照(包括没有已知不表达目标蛋白的初级抗体或组织)以及阳性对照(包括已知表达目标蛋白的组织)15.IHC抗体的选择基于其物种特异性(它们与感兴趣的抗原发生反应的可能性)和使用4、5、6的抗原抗体结合检测系统 ,7.在免疫过氧化物酶的情况下,反应的颜色是由沉淀的色原的选择决定的,通常是二氨基苯胺(棕色)16。另一方面,i mmunofluoresence 利用与荧光团结合的抗体来可视化冷冻组织部分的蛋白质表达,并可轻松分析与染色体检测系统相及的多种蛋白质5,7.
在免疫过氧化物酶技术中,二次抗体与生物蛋白结合,生物锡是一种链接分子,能够招募致色报告分子[avid-生物蛋白复合物(ABC)],导致染色信号的放大。使用ABC报告器方法,过氧化物酶与3,3′-二氨基苯甲酸酯(DAB)发生反应,产生强烈的棕色染色,酶与二级抗体结合,然后用普通光学显微镜进行分析。ABC染色,由于阿维丁对生物锡的高亲和力,产生快速和最佳的反应,很少有二级抗体附着在原抗体反应的部位。这种色谱检测方法允许对信号进行密度分析,提供基于棕色信号水平与蛋白质表达水平18相关性的半定量数据。
使用免疫荧光技术,由于不同荧光铬能够以独特的波长发出光,可以同时检测多种蛋白质,但仔细选择荧光铬以尽量减少光谱重叠非常重要。5.此外,在不同宿主物种中使用原抗体可最大限度地减少交叉反应方面的困难。在这种情况下,每个物种特异性的二级抗体只识别一种类型的原发抗体。荧光记者是小有机分子,包括商业衍生物,如Alexa氟染料。
许多动物模型用于了解特定的生理和病理条件。迄今为止,已经确定许多代谢途径在进化过程中被保存。因此,在斑马鱼等模型生物体上的IHC研究,可以深入了解病理和非病理状况的起源和维持17。本报告旨在说明可在成年斑马鱼组织上执行的IHC协议,用于获取OXA、OX-2R和CB1R在外围和中央层面的分布和定位的详细图像。报告还报告了在成年斑马鱼的周围和中心组织中应用两种主要IHC间接方法的协议。描述是间接方法,它允许在二级抗体与荧光染料(免疫荧光法)或酶报告器(免疫过氧化物酶方法)结合的情况下进行信号扩增。色化检测和荧光检测方法各有优缺点。该协议中报告是在成年斑马鱼中使用IHC,主要是免疫荧光,一种动物模型,广泛用于研究在不同生理和病理条件下进化保存的系统。
Protocol
1. 免疫过氧化物酶方案 注:斑马鱼是由Omid Safari教授(伊朗马什哈德马什哈德费尔多西大学自然资源和环境系渔业系)获得。 组织解剖 通过浸入冰水中牺牲斑马鱼(5部分冰/1部分水,4°C);离开他们,直到停止所有运动,以确保死亡的缺氧。 使用以下解剖方法快速切除肠道和大脑: 将鱼擦干,放在纸巾上,然后放在解剖垫上,阻塞?…
Representative Results
免疫过氧化物酶染色的代表性数据如图1和图2所示。 成人斑马鱼肠道OX-A和OX-2R分布的免疫组织化学分析表明,OX-A和OX-2R的定位位点不同,在DIO斑马鱼肠道细胞中表达增加。在内肠和前肠的细胞中观察到OX-A的强烈棕色染色(图1A,A1)。OX-A的免疫表达在不同的肠道组成中发出明确的信号,从前向内肠下降(图1B,B1)。<strong class=…
Discussion
样品制备
样品制备是IHC的第一个关键步骤。可靠的协议允许维持细胞形态、组织架构和抗原性。此步骤需要正确的组织收集、固定和切片22、23。固定的目的是保存组织,减少组织酶或微生物的作用。特别是,固定步骤可保留细胞成分和生物分子,防止细胞成分(如抗原和酶)的自解和转移,稳定细胞材料免受以下程序的?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项研究得到了桑尼奥大学2015-2016年丰迪·赖斯尔卡·迪阿特内奥(FRA)的支持。
Materials
| Anti CB1 | Abcam | ab23703 | |
| Anti OX-2R | Santa Cruz | sc-8074 | |
| Anti-OXA | Santa Cruz | sc8070 | |
| Aquatex | Merck | 1,085,620,050 | |
| Biotinylated rabbit anti-goat | Vector Lab | BA-5000 | |
| citric acid | Sigma Aldrich | 251275 | |
| Confocal microscope | Nikon | Nikon Eclipse Ti2 | |
| Cryostat | Leica Biosystem | CM3050S | |
| DAPI | Sigma Aldrich | 32670 | |
| Digital Camera | Leica Biosystem | DFC320 | |
| Digital Camera for confocal microscope | Nikon | DS-Qi2 | |
| Donkey anti goat Alexa fluor 488-conjugated secondary antibodies | Thermo Fisher | A11055 | |
| Donkey anti goat Alexa fluor 594-conjugated secondary antibodies | Thermo Fisher | A11058 | |
| Donkey anti rabbit Alexa fluor 488-conjugated secondary antibodies | Thermo Fisher | A21206 | |
| Donkey anti rabbit Alexa fluor 594-conjugated secondary antibodies | Thermo Fisher | A21207 | |
| Ethanol absolute | VWR | 20,821,330 | |
| Frozen section compound | Leica Biosystem | FSC 22 Frozen Section Media | |
| H2O2 | Sigma Aldrich | 31642 | |
| HCl | VWR | 20,252,290 | |
| ImmPACT DAB | Vector lab | SK4105 | |
| Microscope | Leica Biosystem | DMI6000 | |
| Microtome | Leica Biosystem | RM2125RT | |
| Na2HPO4 | Sigma Aldrich | S9763 | |
| NaCl | Sigma Aldrich | S7653 | |
| NaH2PO4H2O | Sigma Aldrich | S9638 | |
| NaOH | Sigma Aldrich | S8045 | |
| Normal Donkey Serum | Sigma Aldrich | D9663 | |
| Normal Rabbit Serum | Vector lab | S-5000 | |
| paraffin wax | Carlo Erba | 46793801 | |
| Paraphormaldeyde | Sigma Aldrich | P6148 | |
| sodium citrate dihydrate | Sigma Aldrich | W302600 | |
| Triton X-100 | Fluka Analytical | 93420 | |
| Trizma | Sigma Aldrich | T1503 | |
| VectaStain Elite ABC Kit | Vector lab | PK6100 | |
| Xylene Pure | Carlo Erba | 392603 |
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Cite This Article
Imperatore, R., D’Angelo, L., De Girolamo, P., Cristino, L., Paolucci, M. Identification of Orexin and Endocannabinoid Receptors in Adult Zebrafish Using Immunoperoxidase and Immunofluorescence Methods. J. Vis. Exp. (148), e59308, doi:10.3791/59308 (2019).