基于甲醇的全支架制备用于视网膜神经节细胞的研究
Summary
甲醇可作为视网膜全膜制剂和长期储存的辅助固定培养基，可用于视网膜神经节细胞的研究。
Abstract
视网膜神经节细胞（RGC）是视网膜的投射神经元，将外部视觉信息传播到大脑。RGCs的病理变化与许多视网膜退行性疾病密切相关。全卡口视网膜免疫染色经常用于RGC的实验研究，以评估视网膜的发育和病理状况。在某些情况下，一些有价值的视网膜样本，例如来自转基因小鼠的样本，可能需要长期保留，而不会影响RGC的形态或数量。为了获得可信和可重复的实验结果，使用有效的保存介质至关重要。在这里，我们描述了甲醇作为辅助固定培养基的作用，用于视网膜整体安装制备和长期储存。简而言之，在分离过程中，将冷甲醇（-20°C）移液到视网膜表面以帮助固定组织并促进其通透性，然后将视网膜储存在冷甲醇（-20°C）中，然后进行免疫染色。该协议描述了视网膜分离工作流程和组织样本储存协议，这对于RGC的研究是有用和实用的。
Introduction
视网膜神经节细胞（RGC）是视网膜中唯一的投射神经元，它们整合并将外部视觉信息传递给大脑1。许多神经退行性疾病，如青光眼和创伤性视神经病变，其特征是RGCs2，3的不可逆转的损伤和丧失。分析RGCs的形态和数量变化是确定神经退行性疾病如何发展和进展的关键步骤4，5。
间接免疫荧光测定是一种广泛接受的监测蛋白质分布和细胞计数的方法。在实验室中，全卡口视网膜免疫染色通常用于RGCs的实验研究，以评估视网膜的生理和病理状况6。用于整个视网膜中RGC定量的最常见标志物包括Brn3a，具有多次剪接的RNA结合蛋白（RBPMS）等7，8。表征RGC的数量和分布需要高质量的全安装视网膜免疫染色。通常，在免疫染色方案中，视网膜在抗体中孵育之前浸入化学固定剂中。理想的固定剂不应改变细胞的形状、抗体表位的可及性或亲和力或组织的线性尺寸9，10。
由于视网膜结构复杂，在制作全贴片视网膜贴片时容易出现视网膜脆性和折叠等问题，以及细胞收缩和细胞核不清晰等一些常见困难，这对实验研究产生了负面影响。此外，并非所有视网膜都会立即进行免疫染色，特别是当涉及到价格昂贵且来源珍贵的转基因小鼠的视网膜时，需要保存额外的视网膜样本以供进一步使用。
适当的固定液可以快速固定组织，避免组织自溶，保持组织细胞的正常形态和结构，并保持蛋白质和其他物质的抗原性10。目前，基于甲醛的固定已广泛应用于各种组织，包括分离的视网膜，半切的眼罩和整个眼球10。组织收缩和细胞形态改变是浸泡在甲醛11中后遇到的两个关键挑战。此外，改良的固定配方正在越来越多地出现，以最大限度地保留视网膜和靶细胞的原始特性9，10。不同的视网膜固定治疗对视网膜结构、蛋白质免疫原性、荧光激发和衰减猝灭周期的影响可能不同12，13。与用福尔马林固定的视网膜相比，用戴维森溶液固定的视网膜在形态上更完整，但戴维森溶液与某些抗体的相容性较差，例如小胶质细胞标记电离钙结合接头分子112。考虑到视网膜的脆弱性质，研究人员自然会想知道视网膜完整性以及靶细胞的性质和形态在长期储存后是否会发生变化。然而，固定液在储存数月后对视网膜和RGC细胞形态的可能影响很少有报道。视网膜固定的优化对于RGC的评估和保存至关重要。
我们详细介绍了一种可靠且技术上简单的方法，该方法用于全安装鼠视网膜染色。我们的方法强调为RGC研究正确准备和储存视网膜，同时考虑到视网膜组织长期储存的需要以及荧光团形成或降解的特定方面。
Protocol
Representative Results
Discussion
固定是保护视网膜的重要步骤，这可能会影响任何基于形态的后续RGC研究。成功的固定可以快速捕获视网膜在暴露于固定介质时的结构和状态，这对于进一步分析至关重要。尽管甲醛被认为是组织和细胞固定和保存中最常见的固定剂之一，但对于某些研究而言，甲醛本身并不总是作为最佳化学固定剂起作用10，14。不同种类的固定剂具有不同的沉淀氨基?…
Declarações
The authors have nothing to disclose.
Acknowledgements
这项工作由湖北省重点实验室开放项目（批准号：2021KFY055）、湖北省自然科学基金（批准号：2020CFB240）和中央高校基本科研业务费（批准号：2042020kf0065）资助。
Materials
| 24-well cell culture cluster | Costar | Eyeball fixation | |
| 24-well hemagglutination plate | Labedit Company | Incubation antibody | |
| Adhesion microscope slides | Citotest | Or similar | |
| Anti-fluorescent quenching mountant | Servicebio | G1401 | Slow down fluorescence quenching |
| BSA (bovine serum albumin) | Servicebio | GC305010 | Blocking reagent |
| Confocal microscope | OLYMPUS | Apply 40x objective lens | |
| Curved scissors | Jiangsu Kanghua Medical Equipment Co., Ltd. | Dissecting tools | |
| Dissecting microscope | RWD Life science Co.,LTD | 77001S | Dissecting tools |
| Forceps | Jiangsu Kanghua Medical Equipment Co., Ltd. | Dissecting tools | |
| Methanol | Sinopharm Chemical Reagent Co., Ltd. | 20210624 | GC≥99.5% |
| Nail polish | SecheVite | Sealing agent | |
| Needles | Shanghai Kindly Enterprises Development Group Co., Ltd. | Accelerate the fixation | |
| Paraformaldehyde solution | Servicebio | G1101 | Eyeball fixation |
| PBS (phosphate buffered saline pH 7.4) | Servicebio | G0002 | Rinse the eyeball |
| Primary antibody: guinea pig anti-RNA-binding protein with multiple splicing (RBPMS) | PhosphoSolutions | Cat. #1832-RBPMS | For immunofluorescence. Used at 1:400 |
| Secondary antibody: Cy3 affiniPure donkey anti-guinea pig IgG (H+L) | Jackson ImmunoResearch | 706-165-148 | For immunofluorescence. Used at 1:400 |
| Straight scissors | Jiangsu Kanghua Medical Equipment Co., Ltd. | Dissecting tools |
Referências
- Sanes, J. R., Masland, R. H. The types of retinal ganglion cells: Current status and implications for neuronal classification. Annual Review of Neuroscience. 38, 221-246 (2015).
- Almasieh, M., Wilson, A. M., Morquette, B., Cueva Vargas, J. L., Di Polo, A. The molecular basis of retinal ganglion cell death in glaucoma. Progress in Retinal and Eye Research. 31 (2), 152-181 (2012).
- Au, N. P. B., Ma, C. H. E. Neuroinflammation, microglia and implications for retinal ganglion cell survival and axon regeneration in traumatic optic neuropathy. Frontiers in Immunology. 13, 860070 (2022).
- Pavlidis, M., Stupp, T., Naskar, R., Cengiz, C., Thanos, S. Retinal ganglion cells resistant to advanced glaucoma: A postmortem study of human retinas with the carbocyanine dye DiI. Investigative Ophthalmology & Visual Science. 44 (12), 5196-5205 (2003).
- Vidal-Sanz, M., et al. Understanding glaucomatous damage: anatomical and functional data from ocular hypertensive rodent retinas. Progress in Retinal and Eye Research. 31 (1), 1-27 (2012).
- Kole, C., et al. Activating transcription factor 3 (ATF3) protects retinal ganglion cells and promotes functional preservation after optic nerve crush. Investigative Ophthalmology & Visual Science. 61 (2), 31 (2020).
- Nadal-Nicolás, F. M., et al. Brn3a as a marker of retinal ganglion cells: qualitative and quantitative time course studies in naive and optic nerve-injured retinas. Investigative Ophthalmology & Visual Science. 50 (8), 3860-3868 (2009).
- Kwong, J. M., Caprioli, J., Piri, N. RNA binding protein with multiple splicing: A new marker for retinal ganglion cells. Investigative Ophthalmology & Visual Science. 51 (2), 1052-1058 (2010).
- Stradleigh, T. W., Ishida, A. T. Fixation strategies for retinal immunohistochemistry. Progress in Retinal and Eye Research. 48, 181-202 (2015).
- Stradleigh, T. W., Greenberg, K. P., Partida, G. J., Pham, A., Ishida, A. T. Moniliform deformation of retinal ganglion cells by formaldehyde-based fixatives. Journal of Comparative Neurology. 523 (4), 545-564 (2015).
- Bucher, D., Scholz, M., Stetter, M., Obermayer, K., Pflüger, H. J. Correction methods for three-dimensional reconstructions from confocal images: I. Tissue shrinking and axial scaling. Journal of Neuroscience Methods. 100 (1-2), 135-143 (2000).
- Chidlow, G., Daymon, M., Wood, J. P., Casson, R. J. Localization of a wide-ranging panel of antigens in the rat retina by immunohistochemistry: Comparison of Davidson’s solution and formalin as fixatives. Journal of Histochemistry & Cytochemistry. 59 (10), 884-898 (2011).
- Tokuda, K., et al. Optimization of fixative solution for retinal morphology: A comparison with Davidson’s fixative and other fixation solutions. Japanese Journal of Ophthalmology. 62 (4), 481-490 (2018).
- Miki, M., Ohishi, N., Nakamura, E., Furumi, A., Mizuhashi, F. Improved fixation of the whole bodies of fish by a double-fixation method with formalin solution and Bouin’s fluid or Davidson’s fluid. Journal of Toxicologic Pathology. 31 (3), 201-206 (2018).
- Zanini, C., Gerbaudo, E., Ercole, E., Vendramin, A., Forni, M. Evaluation of two commercial and three home-made fixatives for the substitution of formalin: A formaldehyde-free laboratory is possible. Environmental Health. 11, 59 (2012).
- Tang, M., et al. An optimized method to visualize the goblet cell-associated antigen passages and identify goblet cells in the intestine, conjunctiva, and airway. Immunobiology. 227 (6), 152260 (2022).
- Brock, R., Hamelers, I. H., Jovin, T. M. Comparison of fixation protocols for adherent cultured cells applied to a GFP fusion protein of the epidermal growth factor receptor. Cytometry. 35 (4), 353-362 (1999).
- Baykal, B., Korkmaz, C., Kocabiyik, N., Ceylan, O. M. The influence of post-fixation on visualising vimentin in the retina using immunofluorescence method. Folia Morphologica. 77 (2), 246-252 (2018).
- Powner, M. B., et al. Visualization of gene expression in whole mouse retina by in situ hybridization. Nature Protocols. 7 (6), 1086-1096 (2012).
- Zhang, N., Cao, W., He, X., Xing, Y., Yang, N. Using methanol to preserve retinas for immunostaining. Clinical and Experimental Ophthalmology. 50 (3), 325-333 (2022).
- Kalesnykas, G., et al. Retinal ganglion cell morphology after optic nerve crush and experimental glaucoma. Investigative Ophthalmology & Visual Science. 53 (7), 3847-3857 (2012).
- Parrilla-Reverter, G., et al. Time-course of the retinal nerve fibre layer degeneration after complete intra-orbital optic nerve transection or crush: a comparative study. Vision Research. 49 (23), 2808-2825 (2009).
