视网膜低温切片、全支架和低渗隔离血管毛细血管微血管组织免疫组化的制备
Summary
我们展示了三种不同的组织制备技术的免疫组化显示大鼠视网膜微血管毛细血管,即低温切片, 全支架, 和低渗的隔离血管网络。
Abstract
视网膜毛细血管在许多眼部疾病中起着重要作用。视网膜血管和微血管毛细血管的免疫组化染色技术是眼科研究的中心。选择合适的方法来可视化微血管毛细血管是至关重要的。我们用血小板衍生生长因子受体β (PDGFRβ) 和神经/胶质抗原 2 (NG2) 的抗体描述了低温切片、全支架和低渗隔离血管的视网膜微血管周细胞免疫组化染色。这使我们可以突出的优点和缺点, 三组织准备的可视化视网膜微血管毛细血管。冷冻切片提供 transsectional 可视化所有视网膜层, 但只包含少数偶尔的横向削减微血管。全芒提供了整个视网膜血管的概述, 但可视化的微循环可能是麻烦的。低渗隔离提供了一种通过去除神经细胞来可视化整个视网膜血管的方法, 但这使得组织非常脆弱。
Introduction
视网膜毛细血管是许多研究实验室的重点, 因为这些细胞在血管的完整性中起着重要的作用。病理条件如糖尿病视网膜病变1, 缺血2, 青光眼3有血管特征, 涉及毛细血管的功能。毛细血管见于内视网膜毛细血管丛。视网膜中央动脉提供内视网膜分支成两层毛细血管丛。内血管床位于神经节细胞和内核层之间。更深层是更加密集和复杂的, 并且在内部和外部核层数之间被本地化4,5。此外, 视网膜的某些部分还包含第三个称为径向 parapapillary 毛细血管的网络。这些是长, 直毛细血管在神经纤维之间和很少吻合互相或其他二丛6。在毛细管壁内, 毛细血管嵌在基底膜内, 线 abluminal 侧血管内皮细胞。
到目前为止, 没有这些毛细血管的唯一生物标记, 可以区分它们与其他血管细胞。血小板衍生生长因子受体β (PDGFRβ) 和神经/胶质抗原 2 (NG2) 是常用的标记, 既存在于毛细血管, 也包括其他血管细胞。毛细血管的识别由于形态学和蛋白质表达式的周细胞子集的存在而进一步复杂化.目前, 最佳的鉴定依赖于蛋白质标记的组合和周细胞在血管壁的特征定位。我们在这里演示了三种不同的组织制备技术的免疫组化 PDGFRβ/NG2 染色的大鼠视网膜微血管毛细血管,即低温切片, 全支架, 和低渗的隔离血管网络。
随着低温切片, 视网膜和巩膜被切断通过视神经。这使得神经元的所有层状结构可视化。不同的十层视网膜是明显的交换核和轴突/树突结构, 可以可视化的污点, 如苏木精/红/红或荧光核 4 ‘, 6-diamidino 2-苯基吲哚 (DAPI)8。代谢要求不同的层9 , 它提供了一种方法来确定厚度或总缺乏的特定层 (例如, 视网膜神经节细胞的损失是视网膜缺血10的标志之一, 11)。血管是明显的横向切开通过视网膜, 使成为可能分开地研究毛细血管丛在各自视网膜层数12,13。
传统上, 视网膜血管网的研究是在全支架上进行的。随着这种组织的准备, 视网膜被切割和扁平的花状结构。该方法是一种相对较快的组织制备技术, 能突出整体结构的视网膜血管, 因此经常被应用于小鼠视网膜新生血管的研究中。在新生小鼠和大鼠视网膜14、15、16、17、18中, 还报道了全装视网膜微血管的成功可视化19. 这些研究显示, 与新生儿视网膜14相比, 成年人的无毛细血管面积更明确的 pericytic 活动。
另一种可视化的方法是在低渗隔离后的视网膜微血管。这种组织制备技术导致视网膜血管和毛细血管被释放的神经细胞。这种类型的孤立视网膜血管网络的二维成像通常是在视网膜胰蛋白酶消化20后进行的, 用于评估糖尿病视网膜病变的血管异常, 包括周细胞丢失和毛细血管退化20,21,22。低渗隔离法提供了对视网膜血管基因和蛋白质调控反应的研究, 因为它们已经做了 rt-pcr 和西方印迹23,24,25。我们提供了一个协议, 以自由浮动的免疫组化染色的低渗隔离视网膜血管作为胰蛋白酶消化的替代品, 以检查微血管毛细血管。
Protocol
Representative Results
Discussion
我们提出三种视网膜制备技术, 可用于微血管性视网膜毛细血管的研究。下面, 我们提供了两种方法之间的比较, 并突出显示了协议中的关键步骤。
随着低温切片, 视网膜被切割成矢状切片, 因此, 有可能获得来自同一视网膜的大量标本。这个方法产生的数字部分使它成为抗体特异性和滴定测试的理想选择, 因为它可以防止不必要的动物牺牲。筹备工作对取得好成绩是决定性的。…
Offenlegungen
The authors have nothing to disclose.
Acknowledgements
这项研究由丹麦 Lundbeck 基金会资助。
Materials
| Geletin from porcine skin | Sigma-Aldrich | G2625-500G | |
| Albumin from chicken egg white | Sigma-Aldrich | A5253-500G | |
| Deoxyribonuclease (DNAse) I from bovine pancreas | Sigma-Aldrich | D5025-15KU | Dissolved in 0.15 M NaCl |
| Bovine serum albumin (BSA) | VWR | 0332-100G | |
| Normal donkey serum | Jackson ImmunoResearch | 017-000-121, lot 129348 | |
| Rabbit anti-PDGFRβ | Santa Cruz | sc-432 | 1:100 |
| Mouse anti-NG2 | Abcam | ab50009 | 1:500 |
| Alexa Fluor 594 AffiniPure Donkey Anti-Rabbit IgG | Jackson ImmunoResearch | 711-585-152 | 1:100 |
| Fluorescein (FITC) AffiniPure Donkey Anti-Mouse IgG (H+L) | Jackson ImmunoResearch | 715-095-151 | 1:100 |
| Cy2 AffiniPure Donkey Anti-Rabbit IgG (H+L) | Jackson ImmunoResearch | 711-225-152 | 1:100 |
| Cy3 AffiniPure Donkey Anti-Mouse IgG (H+L) | Jackson ImmunoResearch | 715-165-150 | 1:100 |
| 4',6-diamidino-2-phenylindole (DAPI) | Sigma-Aldrich | D9542-1MG | Dissolved in DMSO |
| Anti-fading mounting medium | Vector Laboratories | H-1000 | |
| Anti-fading mounting medium with DAPI | Vector Laboratories | H-1200 | |
| Nunc Lab-Tek II 4-well chamber slide | Thermo Fisher Scientific | 154526 |
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