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
<ol>
	<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...

<ol>
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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil-extracellular+traps,+cell-free+DNA+and+immunothrombosis+in+companion+animals:+A+review.">Neutrophil-extracellular traps, cell-free DNA and immunothrombosis in companion animals: A review.</a> Veterinary Pathology. , 300985819861721 (2019).</li><li>de Boer, O. J., Li, X., Goebel, H., van der Wal, A. 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H. L., Tablin, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+Vitro+Canine+Neutrophil+Extracellular+Trap+Formation:+Dynamic+and+Quantitative+Analysis+by+Fluorescence+Microscopy.">In Vitro Canine Neutrophil Extracellular Trap Formation: Dynamic and Quantitative Analysis by Fluorescence Microscopy.</a> Journal of Visualized Experiments. (138), e58083 (2018).</li><li>de Boer, O. J., Li, X., Goebel, H., van der Wal, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+smears+observed+in+H&amp;E-stained+thrombus+sections+are+neutrophil+extracellular+traps.">Nuclear smears observed in H&amp;E-stained thrombus sections are neutrophil extracellular traps.</a> Journal of Clinical Pathology. 69 (2), 181-182 (2016).</li><li>Farkas, &. #. 1. 9. 3. ;. 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F. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+Extracellular+Traps+in+Arterial+and+Venous+Thrombosis.">Neutrophil Extracellular Traps in Arterial and Venous Thrombosis.</a> Seminars in Thrombosis and Hemostasis. 45 (1), 86-93 (2019).</li><li>Li, R. H. L., Johnson, L. R., Kohen, C., Tablin, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+approach+to+identifying+and+quantifying+neutrophil+extracellular+trap+formation+in+septic+dogs+using+immunofluorescence+microscopy.">A novel approach to identifying and quantifying neutrophil extracellular trap formation in septic dogs using immunofluorescence microscopy.</a> BMC Veterinary Research. 14 (1), 210 (2018).</li><li>Brinkmann, V., Abu Abed, U., Goosmann, C., Zychlinsky, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunodetection+of+NETs+in+Paraffin-Embedded+Tissue.">Immunodetection of NETs in Paraffin-Embedded Tissue.</a> Frontiers in Immunology. 7, 513 (2016).</li><li>Moelans, C. B., Oostenrijk, D., Moons, M. J., van Diest, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formaldehyde+substitute+fixatives:+effects+on+nucleic+acid+preservation.">Formaldehyde substitute fixatives: effects on nucleic acid preservation.</a> Journal of Clinical Pathology. 64 (11), 960-967 (2011).</li><li>Rait, V. K., Xu, L., O'Leary, T. J., Mason, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+formalin+fixation+and+antigen+retrieval+with+bovine+pancreatic+RNase+A+II.+Interrelationship+of+cross-linking,+immunoreactivity,+and+heat+treatment.">Modeling formalin fixation and antigen retrieval with bovine pancreatic RNase A II. Interrelationship of cross-linking, immunoreactivity, and heat treatment.</a> Laboratory Investigation: A Journal of Technical Methods and Pathology. 84 (3), 300-306 (2004).</li><li>Willingham, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+alternative+fixation-processing+method+for+preembedding+ultrastructural+immunocytochemistry+of+cytoplasmic+antigens:+the+GBS+(glutaraldehyde-borohydride-saponin)+procedure.">An alternative fixation-processing method for preembedding ultrastructural immunocytochemistry of cytoplasmic antigens: the GBS (glutaraldehyde-borohydride-saponin) procedure.</a> The Journal of Histochemistry and Cytochemistry: Official Journal of the Histochemistry Society. 31 (6), 791-798 (1983).</li><li>Davis, A. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterizing+and+Diminishing+Autofluorescence+in+Formalin-fixed+Paraffin-embedded+Human+Respiratory+Tissue.">Characterizing and Diminishing Autofluorescence in Formalin-fixed Paraffin-embedded Human Respiratory Tissue.</a> The Journal of Histochemistry and Cytochemistry: Official Journal of the Histochemistry Society. 62 (6), 405-423 (2014).</li><li>Banerjee, B., Miedema, B. E., Chandrasekhar, H. 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The authors have nothing to disclose.

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.

<table>
	<tbody>
		<tr>
			<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>
	</tbody>
</table>

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 ...

This protocol of the parafinization followed by antigen retrieval of paraffin-embedded aortic sections can be a useful tool to study the role of neutrophilic tricellular traps in feline thrombosis. Detection of neutrophilic tricellular traps by immunofluorescence in fixed and paraffin-embedded tissue, is superior to conventional histological stents like HNE, as it allows simultaneous detection of safer DNA and extracellular proteins. This protocol can be used in other type of thrombi and in other veterinary species.Identification of neutrophil extracellular traps within feline arterial thrombi suggest that they may play a role in thrombosis in cats. Protocols presented here are guidelines. We strongly encourage investigators to adjust the duration and conditions of the antigen retrieval process to yield a satisfactory signal.To use an automated system to perform deparaffinization and re-hydration of the sections on glass slides, place the glass slides in racks. Submerge completely in 100 percent saline for three minutes. Repeat this step twice.Do not rinse in between steps. Submerge completely in decreasing concentrations of ethanol at room temperature three times for three minutes each. Submerge completely in deionized water for two minutes and repeat one more time.After treatment with TBST, fill the reservoir with deionized water heated to 100 degrees Celsius. Allow the steamer chamber to equilibrate for 20 minutes. On a heat plate, heat the commercially available antigen retrieval solution containing Tris and EDTA at pH 9.0 to 95 to 97 degrees Celsius.Ensure that it does not boil. Suberge the slides completely in the heated antigen retrieval solution and continue the application of external heating via the steamer for 20 minutes. Next, wash the slides three times with TBST for five minutes.Now, place the sections in blocking buffer 1. Incubate for two hours at room temperature under gentle rocking between 30 and 50 rpm. Without washing, immediately apply 100 microliters of diluted rabbit polyclonal anti-human citrullinated histone H3 antibody directly onto the slide.Place a cover slip on each section to allow even distribution of the antibody mixture. Incubate for 12 to 16 hours at four degrees Celsius with gentle rocking. After that, wash the slides three times with TBST for five minutes each time.To apply antibody, use a pipet and evenly distribute 100 microliters of goat anti-rabbit antibody conjugated to Alexa Fluor 488. Cover the slides with tin foil and incubate the slides for one hour at room temperature under gentle rocking. After washing with TBST according to the manuscript, incubate the sections in blocking buffer 2 overnight at four degrees celsius under gentle rocking.Protect from the light. Wash with TBST three times for five minutes each time. Block the sections in blocking buffer 3 as done previously at room temperature for two hours under gentle rocking.Incubate the sections with biotin laded ployclonal rabbit anti-human neutrophil elastase antibody at four degree Celsius for 12 to 16 hours as described previously. After washing with TBST incubate the slides with Alexa Fluor 594 Streptavidin Conjugate for one hour at room temperature. After that, wash with TBST one time for five minutes.Apply 100 microliters of Autofluorescense Quenching solution mixture directly onto the sections for one minute as instructed by the manufacturer. Immediately wash the slides with TBST six times for 10 minutes each time. Cover each slide with 100 microliters of 300 nanomolar DAPI for five minutes in the dark.Then wash with TBST five times for three minutes each time. Apply a drop of antifade mounting medium directly onto the glass slide surrounding the section. Place a cover slip gently onto the section without creating any bubbles.Allow the samples to cure overnight in the dark at four degrees Celsius. To locate thrombi, scan cranially to caudally along the length of the aorta, aortic bifurcation and each femeral artery using phase contrast microscopy with a 10x objective. First examine the sections for cell-free DNA using the DAPI channel with the excitation at 357 and 44 nanometers.Identify extracellular neutrophil elastase and citrullinated histone H3 on the Texas red and green fluorescent protein channels respectively. Maintain consistent exposure time and gains of each channel throughout the acquisition of images to avoid saturation in pixel intensity. Neutrophil extracellular traps are identified based co-localization of neutrophil elastase, citrullinated histone H3 and cell-free DNA.Using hematoxylin and eosin stain, and brightfield microscopy, feline arterial thrombi consist of red blood cells, leukocytes, fibrin and platelets. NETs appeared as networks of deep purple threads of various lengths surrounding nearby erythrocytes and leukocytes. Using this protocol, immunofluorescence microscopy revealed large aggregates of NETs composed of cell-free DNA, extracellular citrullinated histone H3 and neutrophil elastase.Under phase contrast in immune microscopy, a thrombus was characterized as a well-demarcated structure within the vascular space. However, thrombi were not detected in any of the control samples. This figure demonstrates profound autofluoresence of clot elements like erythrocytes when imaged at the 488 nanometer wavelength.Brief autofluoresence quenching after immunolabelling significantly increased the sensitivity of protein co-localization and NET detection even in areas with an abundance of erythrocytes. The duration of fixation effects the immunoreactivity of certain antigens. We recommend fixation for no longer than 24 hours par the dehydration and paraffin embedding.Alternatively, methanol-free paraformaldehyde can be used to limit artifact by formic acid and ketones from oxidation of formaldehyde. To prevent interference when using primary antibodies originating from the same species, we included additional blocking step to saturate any remaining binding sites on the secondary antibodies. Investigators must include negative controls consisting of isotype antibodies, exclusion of either primary antibodies and immuno-labeling step and biological controls from cats without thrombosis.Autofluoresence of tissue limits of thrombi can hammer the microscopic identification of neutrophil extracellular traps. Investigator can minimize the fluorescence by the use of far-red fluorophores. This technique can be a valuable tool for the study of neutrophil extracellular traps in other veterinary species.This can provide a better understanding of the pathophysiology of thrombosis.

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7.9K Görüntüleme.  University of California, Davis. Parafinizasyon bu protokol parafinin gömülü aort kesitlerinin antijen alımı takip kedi trombozu nötrofilik trisellüler tuzakların rolünü incelemek için yararlı bir araç olabilir. Sabit ve parafin gömülü dokuda immünororesans tarafından nötrofilik trisellüler tuzakların saptanması, daha güvenli DNA ve ekstrasellüler proteinlerin eşzamanlı olarak algılanmasına olanak sağladığından HNE gibi konvansiyonel histolojik stentlerden üstündür. Bu protokol diğer tromb...