小鼠视网膜冷冻节的制备,用于免疫性植物化学
Summary
本报告介绍了制备冷冻小鼠视网膜部分用于免疫组织化学(IHC)的综合方法。 所述方法包括眼部后杯的解剖、甲醛固定、嵌入最佳切割温度(OCT)介质和组织定向、切片和免疫染色。
Abstract
为免疫组织化学 (IHC) 制备高质量的小鼠眼部部分对于评估视网膜结构和功能以及确定视网膜疾病背后的机制至关重要。在整个组织制备过程中保持结构完整性对于获得可重复的视网膜 IHC 数据至关重要,但由于视网膜细胞结构的脆弱性和复杂性,这些数据可能具有挑战性。强固定剂,如10%形式蛋白或布恩的溶液,以最佳方式保持视网膜结构,它们通常通过增强背景荧光和/或减少抗体-表位相互作用(称为表位掩蔽的过程)来阻碍IHC分析。另一方面,较温和的固定剂,如4%的甲醛,减少背景荧光和表位遮蔽,必须采用细致的解剖技术来保持视网膜结构。在本文中,我们将介绍一种为 IHC 制备小鼠眼后杯的综合方法,该方法足以保留大多数抗体-表皮相互作用,而不会损失视网膜结构完整性。我们包括具有代表性的IHC与抗体的各种视网膜细胞类型标记,以说明组织保存和方向在最佳和次优条件下。我们的目标是通过提供从眼后杯解剖到IHC的完整方案来优化视网膜的IHC研究。
Introduction
免疫组织化学(IHC)是一种强大的技术,用于在原位1、2、3定位组织的特定蛋白质和细胞结构。不适当的固定方法和复杂组织的次优切片可能会破坏组织结构,产生高背景染色或减少抗体-表皮相互作用,导致染色伪影,进而造成对IHC 数据4.由于脊椎动物视网膜是一种复杂且高度组织的神经器官,由相互连接的光受体、内神经细胞和神经细胞层组成,因此非常脆弱,在解剖和切片过程中很容易被破坏。从小鼠眼部解剖和定向到免疫染色的详细、标准化和经过验证的协议将有助于显著减少 IHC 伪影,从而提高结果的可靠性,并允许获得更准确的比较数据分析。
IHC的组织制备有许多方案,但是,并非所有方案都适合视网膜组织。强固定剂,如10%正式或布因的解决方案保留视网膜结构在解剖和切片5。不幸的是,由于表位6的化学修饰,强固定剂往往导致增强的背景荧光和表位掩蔽。另一方面,温和的固定剂,如4%的甲醛(PFA)可以减轻其中一些伪影,但需要细致的解剖和切片,以保持最佳的视网膜结构。PFA 迅速穿透组织,但交叉链接蛋白质非常缓慢,降低了表位掩蔽的风险。由于短时间的PFA孵育是一种相对温和的固定,组织往往需要快速冷冻,以保持抗原。在组织冻结期间避免冰晶形成是很重要的,因为它们会扭曲和损害细胞和组织的完整性7。
在这里,我们描述了用于小鼠眼后杯解剖、固定和冷冻保护的详细和标准化协议,这些电池杯可生成一致可靠的 IHC 数据。
Protocol
Representative Results
Discussion
由于小鼠眼睛的体积和形状小,视网膜易碎,小鼠视网膜解剖是一个微妙的过程。尽管进行高质量的解剖是一个实践问题,但拥有详细的协议,提供有效的方法和提示是获得视网膜部分和IHC的必要条件。除了此处介绍的协议外,还有几个提示允许一致的高质量视网膜部分,适用于可重复的 IHC。
在开始解剖之前,重要的是要舒适,放松,并设置房间照明,以在小鼠眼睛微解剖的最佳视力。为…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到了NIH(RO1-GMO97327)和大学研究基金会(UPenn)的资金支持。我们特别感谢斯韦特兰娜·萨维娜在开发免疫组织化学方案方面给予的帮助,戈登·鲁塞尔(宾夕法尼亚大学)和宾夕法尼亚大学Vet成像核心中心为显微镜和莱斯利·金博士提供了显微镜和莱斯利·金博士的辅助,帮助她阅读了这份手稿.
Materials
| 6qt. Stainless Steel Beaker (185 x 218mm) | Gilson | #MA-48 | For liquid nitrogen (Figure 1E) |
| Aluminum foil | |||
| Cauterizer | Bovie | #AA01 | To mark eye orientation (Figure 1A) |
| Curved forceps, Dumont #5/45 tweezers, 45-degrees bent | Electron Microscopy Sciences | #72703-D | For mouse eye enucleation and maintenance (Figure 1A) |
| Curved scissors, Vannas Spring Scissors – 2.5mm Cutting Edge | Fine Science Tools | #15002-08 | To circumferentially cut the cornea (Figure 1B) |
| Dental wax-coated 35mm dissection dish | For eye cup dissection (Figure 1C) | ||
| Dissecting microscope | Leica, Houston, USA | MZ12.5 High-performance stereomicroscope | |
| Micro Knives – Plastic Handle | Fine Science Tools | #10315-12 | To make a small incision at the burn mark (Figure 1A) |
| Pink dental wax | Electron Microscopy Sciences | #72660 | For coating dissection dish |
| Slide Rack and Coverplate | Ted Pella | #36107 | For retinal IHC (Figure 1G) |
| Stainless Steel Beaker (89 x 114mm) | Gilson | #MA-40 | For isopentane bath (Figure 1E) |
| Styrofoam box | For insulated cooler | ||
| Thin forceps Dumont #5 | Fine Science Tools | #11254-20 | To remove the cornea and extract the lens (Figure 1B) |
| Tissue-Tek cryomold (10mm x 10mm x 5mm) | Electron Miscroscopy Sciences | #62534-10 | Mold for OCT embedding |
| Buffers and Reagents | Company | Catalog Number | Comments |
| Blocking solution | 2% Normal Horse Serum, 1.5% Cold Fish skin Gelatin (at 40-50% in H2O, cat #G7765), 5% bovine serum albumin (cat #AK1391-0100) in permeabilization buffer. | ||
| Gelvatol (anti-fading mounting media) | Under the fume hood, dissolve 0.337g DABCO (Sigma cat #D2522-25G) in 10mL Fluoromount G (Fisher cat #OB100-01). Adjust to pH 8-8.5 with 12N HCl (~ 5 drops). Store in amber drop bottle at 4°C (8). | ||
| Hoechst 33342 1000x Stock | Thermo Scientific | #62249 | 1 mg/ml in PBS (1000x). Aliquot and store in dark, at 4°C for up to 1 year or at -20°C for long term storage. Prepare fresh at 1 ug/mL (1x) |
| Isopentane, 99% | GFS Chemicals | #2961 | |
| Liquid Nitrogen | |||
| Paraformaldehyde (PFA) in PBS | Electron Microscopy Sciences | #15710 | Prepared fresh 4% PFA from 16% PFA stock. Store the remaining 16% PFA at 4°C in the dark. |
| Permeabilization solution | PBS + 0.25% Triton-X (AMRESCO cat #0694-1L) + 0.05% NaN3. Prepare fresh. | ||
| Phosphate-buffered saline (PBS) | Bioworld | #41620015-20 | 0.02 g/L KCl, 0.02 g/L KH2PO4, 0.8 g/L NaCl, 0.216 g/L Na2HPO4 , pH 7.4. Prepared from 10x stock |
| Sucrose solutions | Fisher Scientific | #S-5-500 | Dissolve the appropriate amount of sucrose in 37°C PBS (takes ~15 min to dissolve). May be stored for a few days at 4°C. |
| Tissue-Tek OCT-compound | Sakura | #4583 | |
| Antibodies | Company | Catalog Number | Comments |
| anti-calbindin | Sigma-Aldrich | C8666 | To label horinzotal cells |
| anti-GAD65 | Chemicon | AB5082 | To label GABAergic amacrine cells |
| anti-GS | BD Transduction | 610517 | To label Müller cells |
| anti-rhodopsin | Millipore | MAB5316 | To label rods |
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