使用免疫荧光显微镜鉴定帕芬-嵌入式猫动脉血栓中中性粒细胞外陷阱
1Veterinary Medical Teaching Hospital, School of Veterinary Medicine,University of California, Davis, 2Department of Veterinary Surgical and Radiological Sciences, School of Veterinary Medicine,University of California, Davis, 3Department of Veterinary Medicine and Epidemiology, School of Veterinary Medicine,University of California, Davis
Summary
我们描述了一种使用热诱导抗原检索和双免疫标记协议识别甲醛固定和石蜡嵌入的猫科动物心性动脉血栓中嗜中性粒细胞外陷阱（NETs）的方法。
Abstract
中性粒细胞外陷阱（NETs），由无细胞DNA（cfDNA）和蛋白质（如组蛋白和嗜中性粒细胞酶酶（NE）组成，由嗜中性粒细胞释放，以响应全身炎症或病原体。虽然NETs以前曾被证明能增强血栓形成和抑制人类和狗的纤维化，但NET在有心性动脉血栓栓塞（CATE）的猫中的作用，这是一种威胁生命的并发症，仅次于肥大心肌病，未知。一种标准化的方法识别和量化猫有氧动脉血栓中的NET，将增进我们对猫在CAT中病理作用的理解。在这里，我们描述了一种技术，用于识别主动脉分叉内甲醛固定和石蜡嵌入血栓中的NET，在尸检期间提取。与二甲苯脱硫后，主动脉部分进行了间接热诱导抗原检索。然后，通过无细胞DNA（cfDNA）、西特鲁普林语H3（citH3）和中性粒细胞酶（NE）共同定位，利用免疫荧光显微镜，发现部分阻塞、渗透和外游性NETs。为了优化血栓中NET的免疫检测，在显微镜之前使用自动荧光淬火过程限制了组织元素的自荧光。这项技术可能是研究其他物种的NET和血栓形成的有用工具，并为这种复杂情况的病理生理学提供了新的见解。
Introduction
患有肥大心肌病的猫有生命危险血栓栓塞并发症11，2。2尽管与猫科动物心电源性动脉血栓栓塞（CATE）相关的发病率和死亡率很高，但CAT在猫身上的基本病理生理学还知之甚少。也有有限的诊断和治疗工具，以治疗和识别猫在这种毁灭性的条件3的风险。
除了在先天免疫中的作用外，中性粒细胞外嗜中性粒细胞外陷阱（NETs）在血栓形成中发挥了作用，这种陷阱是网络状的无细胞DNA（cfDNA）网络，含有组蛋白和粒状蛋白质，如中性粒细胞酶酶（NE）和骨髓红酶。嗜中性粒细胞在反应全身炎症、直接接触病原体以及与活性血小板44、5、6、75,6,7相互作用时进行神经衰弱的形成。在狗中，中性粒细胞衍生的DNA已被证明能抑制血块性结质，而NET蛋白加速血块形成。NET捕获循环细胞和凝固成分的能力也是其血栓生成特性88、9、10、11、12的关键。,9,10,11,12
通过细胞外嗜中性粒细胞蛋白、组蛋白和cfDNA的共定位检测出NET。因此，通过脱硫素组织的免疫荧光对固定组织中NET的识别和定量优于传统的血氧林和欧辛（H&E）染色，使用明亮的场显微镜44，5。5利用免疫荧光显微镜进行的几项人体研究确定NETs为冠状动脉血栓、脑卒中、动脉炎和静脉血栓13、14、15、16、17的结构成分。13,14,15,16,17迄今为止，尚未描述一种检测和量化猫科动物血栓中NET的标准化方法。由于在猫科动物心性动脉血栓中识别NETs可能有助于未来在NET和血栓的转化研究，我们描述了NET识别和评估在猫的石蜡嵌入动脉血栓的技术。
Protocol
此处描述的所有方法均按照加州大学戴维斯分校机构动物护理和使用委员会的准则执行。在业主同意下进行组织解剖和活检。 1. 组织固定、嵌入和切片 在人道安乐死或死亡后不久，切除主动脉分叉，包括下降的主动脉、股动脉和常见的伊利亚动脉（图1A）。布伦特解剖出筋膜 （图 1B） 之前，它完全浸入 10% 中性缓冲形式，…
Representative Results
利用该协议进行除石蜡化、热诱导抗原检索和石蜡嵌血栓的双重免疫标签，我们首次确定了猫科动物CATE中的NET。主动脉分叉内的血栓通过荧光显微镜和亮场显微镜使用标准H&E染色和相位对比显微镜定位。在明亮的田间显微镜下，猫动脉血栓由红血球、白细胞、纤维蛋白和血小板组成（图3A）。虽然H&E不能染色特定的NET组件，但NET经常作为围绕附近红细胞和白细胞的各种长度?…
Discussion
我们描述了一种协议，使用双免疫标记协议和免疫荧光显微镜识别固定猫科动物心电源动脉血栓中的NET。虽然只有心电动脉血栓被染色，但理论上，这种协议可用于其他类型的血栓和其他兽医物种。猫动脉血栓内NET的识别表明，NET在猫的血栓形成中可能起一定的作用。
在固定和石蜡嵌入组织中通过免疫荧光检测NET优于H&E等传统组织学污渍，后者通常显示由嗜中性粒细胞包围?…
Declarações
The authors have nothing to disclose.
Acknowledgements
这项研究得到了加州大学戴维斯分校动物健康中心（CCAH 2018-30-F）的资金支持。作者要感谢凯文·伍拉德博士使用荧光显微镜。
Materials
| 4,6-Diamidino-2-phenylin (DAPI) | Life Technologies Corporation | D1306 | |
| Alexa Fluor 594 Streptavidin conjugate | ThermoFisher Scientific | Catalog # S11227 | |
| Anti-citrullinated histone H3 antibody | Abcam | Ab5103 | |
| EVOS FL Cell Imaging System | ThermoFisher Scientific | AMEFC4300 | |
| EVOS Imaging System Objective 10x | ThermoFisher Scientific | AMEP4681 | NA 0.25, WD 6.9/7.45 mm |
| EVOS Imaging System Objective 20x | ThermoFisher Scientific | AMEP4682 | NA 0.40, WD 6.8 mm |
| EVOS Imaging System Objective 40x | ThermoFisher Scientific | AMEP4699 | NA 0.75, WD 0.72 mm |
| Goat anti-rabbit Alexa Fluor 488 antibody | ThermoFisher Scientific | Catalog # A32723 | |
| Goat serum | Jackson Immuno Research Labs | Catalog # NC9660079. Manufacturer Part # 005-000-121 | |
| Neutrophil elastase antibody | Bioss Antibodies | Bs-6982R-Biotin | Rabbit polyclonal Antibody, Biotin conjugated |
| NP40 | Pierce | Product # 28324. Lot # EJ64292 | |
| Positive charged microscope slides | Thomas Scientific | Manufacturer No. 1354W-72 | |
| Rabbit serum | Life Technology | Catalog # 10510 | |
| Target Retrieval Solution | Agilent Dako | S2367 | TRIS/EDTA, pH 9 (10x) |
| TrueVIEW Autofluorescence Quenching Kit | Vector Laboratories | SP-8400 |
Referências
- Maron, B. J., Fox, P. R. Hypertrophic cardiomyopathy in man and cats. Journal of Veterinary Cardiology: The Official Journal of the European Society of Veterinary Cardiology. 17, 6-9 (2015).
- Payne, J. R., et al. Prognostic indicators in cats with hypertrophic cardiomyopathy. Journal of Veterinary Internal Medicine. 27 (6), 1427-1436 (2013).
- Borgeat, K., Wright, J., Garrod, O., Payne, J. R., Fuentes, V. L. Arterial Thromboembolism in 250 Cats in General Practice: 2004-2012. Journal of Veterinary Internal Medicine. 28 (1), 102-108 (2014).
- Brinkmann, V., Zychlinsky, A. Beneficial suicide: why neutrophils die to make NETs. Nature Reviews. Microbiology. 5 (8), 577-582 (2007).
- Goggs, R., Jeffery, U., LeVine, D. N., Li, R. H. L. Neutrophil-extracellular traps, cell-free DNA and immunothrombosis in companion animals: A review. Veterinary Pathology. , 300985819861721 (2019).
- de Boer, O. J., Li, X., Goebel, H., van der Wal, A. C. Nuclear smears observed in H & E-stained thrombus sections are neutrophil extracellular traps. Journal of Clinical Pathology. 69 (2), 181-182 (2016).
- Li, R., Tablin, F. A Comparative Review of Neutrophil Extracellular Traps in Sepsis. Frontiers in Veterinary Sciences. 5 (291), (2018).
- Borissoff, J. I., et al. Elevated levels of circulating DNA and chromatin are independently associated with severe coronary atherosclerosis and a prothrombotic state. Arteriosclerosis, Thrombosis, and Vascular Biology. 33 (8), 2032-2040 (2013).
- Moschonas, I. C., Tselepis, A. D. The pathway of neutrophil extracellular traps towards atherosclerosis and thrombosis. Atherosclerosis. 288, 9-16 (2019).
- Perdomo, J., et al. Neutrophil activation and NETosis are the major drivers of thrombosis in heparin-induced thrombocytopenia. Nature Communications. 10 (1), 1322 (2019).
- Li, B., et al. Neutrophil extracellular traps enhance procoagulant activity in patients with oral squamous cell carcinoma. Journal of Cancer Research and Clinical Oncology. 145 (7), 1695-1707 (2019).
- Li, R. H. L., Tablin, F. In Vitro Canine Neutrophil Extracellular Trap Formation: Dynamic and Quantitative Analysis by Fluorescence Microscopy. Journal of Visualized Experiments. (138), e58083 (2018).
- de Boer, O. J., Li, X., Goebel, H., van der Wal, A. C. Nuclear smears observed in H&E-stained thrombus sections are neutrophil extracellular traps. Journal of Clinical Pathology. 69 (2), 181-182 (2016).
- Farkas, &. #. 1. 9. 3. ;. Z., et al. Neutrophil extracellular traps in thrombi retrieved during interventional treatment of ischemic arterial diseases. Thrombosis Research. 175, 46-52 (2019).
- Qi, H., Yang, S., Zhang, L. Neutrophil Extracellular Traps and Endothelial Dysfunction in Atherosclerosis and Thrombosis. Frontiers in Immunology. 8, 928 (2017).
- Laridan, E., et al. Neutrophil extracellular traps in ischemic stroke thrombi. Annals of Neurology. 82 (2), 223-232 (2017).
- Laridan, E., Martinod, K., Meyer, S. F. D. Neutrophil Extracellular Traps in Arterial and Venous Thrombosis. Seminars in Thrombosis and Hemostasis. 45 (1), 86-93 (2019).
- Li, R. H. L., Johnson, L. R., Kohen, C., Tablin, F. A novel approach to identifying and quantifying neutrophil extracellular trap formation in septic dogs using immunofluorescence microscopy. BMC Veterinary Research. 14 (1), 210 (2018).
- Brinkmann, V., Abu Abed, U., Goosmann, C., Zychlinsky, A. Immunodetection of NETs in Paraffin-Embedded Tissue. Frontiers in Immunology. 7, 513 (2016).
- Moelans, C. B., Oostenrijk, D., Moons, M. J., van Diest, P. J. Formaldehyde substitute fixatives: effects on nucleic acid preservation. Journal of Clinical Pathology. 64 (11), 960-967 (2011).
- Rait, V. K., Xu, L., O’Leary, T. J., Mason, J. T. Modeling formalin fixation and antigen retrieval with bovine pancreatic RNase A II. Interrelationship of cross-linking, immunoreactivity, and heat treatment. Laboratory Investigation: A Journal of Technical Methods and Pathology. 84 (3), 300-306 (2004).
- Willingham, M. C. An alternative fixation-processing method for preembedding ultrastructural immunocytochemistry of cytoplasmic antigens: the GBS (glutaraldehyde-borohydride-saponin) procedure. The Journal of Histochemistry and Cytochemistry: Official Journal of the Histochemistry Society. 31 (6), 791-798 (1983).
- Davis, A. S., et al. Characterizing and Diminishing Autofluorescence in Formalin-fixed Paraffin-embedded Human Respiratory Tissue. The Journal of Histochemistry and Cytochemistry: Official Journal of the Histochemistry Society. 62 (6), 405-423 (2014).
- Banerjee, B., Miedema, B. E., Chandrasekhar, H. R. Role of basement membrane collagen and elastin in the autofluorescence spectra of the colon. Journal of Investigative Medicine: The Official Publication of the American Federation for Clinical Research. 47 (6), 326-332 (1999).
- Hirsch, R. E., Zukin, R. S., Nagel, R. L. Intrinsic fluorescence emission of intact oxy hemoglobins. Biochemical and Biophysical Research Communications. 93 (2), 432-439 (1980).
- Billinton, N., Knight, A. W. Seeing the wood through the trees: a review of techniques for distinguishing green fluorescent protein from endogenous autofluorescence. Analytical Biochemistry. 291 (2), 175-197 (2001).
- Mosiman, V. L., Patterson, B. K., Canterero, L., Goolsby, C. L. Reducing cellular autofluorescence in flow cytometry: an in-situ method. Cytometry. 30 (3), 151-156 (1997).
- Ducroux, C., et al. Thrombus Neutrophil Extracellular Traps Content Impair tPA-Induced Thrombolysis in Acute Ischemic Stroke. Stroke. 49 (3), 754-757 (2018).
Citar este artigo
Duler, L., Nguyen, N., Ontiveros, E., Li, R. H. L. Identification of Neutrophil Extracellular Traps in Paraffin-Embedded Feline Arterial Thrombi using Immunofluorescence Microscopy. J. Vis. Exp. (157), e60834, doi:10.3791/60834 (2020).