Name: identification of neutrophil extracellular traps in paraffin-embedded feline arterial thrombi using immunofluorescence microscopy
Uploaded: 2020-03-29T13:00:00
Description: Watch this Scientific Journal Video about Identification of Neutrophil Extracellular Traps in Paraffin-Embedded Feline Arterial Thrombi using Immunofluorescence Microscopy at JoVE.com

The study was supported by funds from the University of California, Davis, Center for Companion Animal Health (CCAH 2018-30-F). The authors would like to acknowledge Dr. Kevin Woolard for usage of the fluorescence microscope.

All methods described here were performed in accordance to the guidelines of the Institutional Animal Care and Use Committee at the University of California, Davis. Necropsies and biopsies of tissues were performed with owners&#8217; consent.
1. Tissue fixation, embedding, and sectioning
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	<li>Dissect out the aortic bifurcation, including the descending aorta, femoral artery, and the common iliac arteries (Figure 1A), shortly after humane euthanasia or death...

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We describe a protocol to identify NETs in fixed feline cardiogenic arterial thrombi using a double immunolabeling protocol and immunofluorescence microscopy. Although only cardiogenic arterial thrombi were stained, in theory this protocol could be used for other types of thrombi and in other veterinary species. Identification of NETs within feline arterial thrombi suggests that NETs may play a role in thrombosis in cats.
Detection of NETs by immunofluorescence in fixed and paraffin-embedded t...

Cats with hypertrophic cardiomyopathy are at risk of life-threatening thromboembolic complications1,2. Despite the high morbidity and mortality associated with feline cardiogenic arterial thromboembolism (CATE), the underlying pathophysiology of CATE in cats is poorly understood. There are also limited diagnostic and therapeutic tools to treat and identify cats at risk of this devastating condition3.
In addition to its role in innate immunity, neutrophils have been shown to play a role in thrombosis by releasing neutrophil extracellular traps (NETs), which are web-like networks of cell-free DNA (cfDNA) encrusted with histones and granular proteins like neutrophil elastase (NE) and myeloperoxidase. Neutrophils undergo NETs formation in response to systemic inflammation, direct encounter with pathogens, and interaction with activated platelets4,5,6,7. In dogs, neutrophil-derived DNA has been shown to inhibit clot lysis, while NET proteins accelerate clot formation. The ability of NETs to trap circulating cells and coagulation components is also key to their thrombogenic properties8,9,10,11,12.
NETs are detected by colocalization of extracellular neutrophil proteins, histones, and cfDNA. Because of this, the identification and quantification of NETs in fixed tissues by immunofluorescence of deparaffinized tissues is superior to traditional hematoxylin and eosin (H&#38;E) stain using bright field microscopy4,5. Several human studies using immunofluorescence microscopy identified NETs as structural components of coronary arterial thrombi, cerebral stroke thrombi, atherothrombosis, and venous thrombi13,14,15,16,17. To date, a standardized method to detect and quantify NETs in feline thrombi has not been described. Because the identification of NETs in feline cardiogenic arterial thrombi may facilitate future translational research in NETs and thrombosis, we describe techniques of NET identification and assessment in paraffin-embedded arterial thrombi in cats.

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			<td>4,6-Diamidino-2-phenylin (DAPI)</td>
			<td>Life Technologies Corporation</td>
			<td>D1306</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 594 Streptavidin conjugate</td>
			<td>ThermoFisher Scientific</td>
			<td>Catalog # S11227</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-citrullinated histone H3 antibody</td>
			<td>Abcam</td>
			<td>Ab5103</td>
			<td></td>
		</tr>
		<tr>
			<td>EVOS FL Cell Imaging System</td>
			<td>ThermoFisher Scientific</td>
			<td>AMEFC4300</td>
			<td></td>
		</tr>
		<tr>
			<td>EVOS Imaging System Objective 10x</td>
			<td>ThermoFisher Scientific</td>
			<td>AMEP4681</td>
			<td>NA 0.25, WD 6.9/7.45 mm</td>
		</tr>
		<tr>
			<td>EVOS Imaging System Objective 20x</td>
			<td>ThermoFisher Scientific</td>
			<td>AMEP4682</td>
			<td>NA 0.40, WD 6.8 mm</td>
		</tr>
		<tr>
			<td>EVOS Imaging System Objective 40x</td>
			<td>ThermoFisher Scientific</td>
			<td>AMEP4699</td>
			<td>NA 0.75, WD 0.72 mm</td>
		</tr>
		<tr>
			<td>Goat anti-rabbit Alexa Fluor 488 antibody</td>
			<td>ThermoFisher Scientific</td>
			<td>Catalog # A32723</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat serum</td>
			<td>Jackson Immuno Research Labs</td>
			<td>Catalog # NC9660079. Manufacturer Part # 005-000-121</td>
			<td></td>
		</tr>
		<tr>
			<td>Neutrophil elastase antibody</td>
			<td>Bioss Antibodies</td>
			<td>Bs-6982R-Biotin</td>
			<td>Rabbit polyclonal Antibody, Biotin conjugated</td>
		</tr>
		<tr>
			<td>NP40</td>
			<td>Pierce</td>
			<td>Product # 28324. Lot # EJ64292</td>
			<td></td>
		</tr>
		<tr>
			<td>Positive charged microscope slides</td>
			<td>Thomas Scientific</td>
			<td>Manufacturer No. 1354W-72</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit serum</td>
			<td>Life Technology</td>
			<td>Catalog # 10510</td>
			<td></td>
		</tr>
		<tr>
			<td>Target Retrieval Solution</td>
			<td>Agilent Dako</td>
			<td>S2367</td>
			<td>TRIS/EDTA, pH 9 (10x)</td>
		</tr>
		<tr>
			<td>TrueVIEW Autofluorescence Quenching Kit</td>
			<td>Vector Laboratories</td>
			<td>SP-8400</td>
			<td></td>
		</tr>
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identification of neutrophil extracellular traps in paraffin-embedded feline arterial thrombi using immunofluorescence microscopy

Neutrophil extracellular traps (NETs), composed of cell-free DNA (cfDNA) and proteins like histones and neutrophil elastase (NE), are released by neutrophils in response to systemic inflammation or pathogens. Although NETs have previously been shown to augment clot formation and inhibit fibrinolysis in humans and dogs, the role of NETs in cats with cardiogenic arterial thromboembolism (CATE), a life-threatening complication secondary to hypertrophic cardiomyopathy, is unknown. A standardized method to identify and quantify NETs in cardiogenic arterial thrombi in cats will advance our understanding of their pathological role in CATE. Here, we describe a technique to identify NETs in formaldehyde-fixed and paraffin-embedded thrombi within the aortic bifurcation, extracted during necropsy. Following deparaffinization with xylene, aortic sections underwent indirect heat-induced antigen retrieval. Sections were then blocked, permeabilized, and ex vivo NETs were identified by colocalization of cell-free DNA (cfDNA), citrullinated histone H3 (citH3), and neutrophil elastase (NE) using immunofluorescence microscopy. To optimize the immunodetection of NETs in thrombi, autofluorescence of tissue elements was limited by using an autofluorescence quenching process prior to microscopy. This technique could be a useful tool to study NETs and thrombosis in other species and offers new insights into the pathophysiology of this complex condition.

Using this protocol for deparaffinization, heat-induced antigen retrieval, and double immunolabeling of paraffin-embedded thrombi, we identified NETs in feline CATE for the first time. Thrombi within the aortic bifurcation were located by fluorescence microscopy and bright field microscopy using standard H&#38;E staining and phase contrast microscopy. On bright field microscopy, feline arterial thrombi consisted of red blood cells, leukocytes, fibrin, and platelets (Figure 3A). Although H&#3...

Watch this Scientific Journal Video about Identification of Neutrophil Extracellular Traps in Paraffin-Embedded Feline Arterial Thrombi using Immunofluorescence Microscopy at JoVE.com

Identification of Neutrophil Extracellular Traps in Paraffin-Embedded Feline Arterial Thrombi using Immunofluorescence Microscopy

We describe a method to identify neutrophil extracellular traps (NETs) in formaldehyde-fixed and paraffin-embedded feline cardiogenic arterial thrombi using heat-induced antigen retrieval and a double immunolabeling protocol.

Neutrophil extracellular traps (NETs), composed of cell-free DNA (cfDNA) and proteins like histones and neutrophil elastase (NE), are released by ...

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