Name: Identification of Neutrophil Extracellular Traps in Paraffin-Embedded Feline Arterial Thrombi using Immunofluorescence Microscopy (Video) | JoVE Journal
Uploaded: 2020-03-29T13:00:00.000+00:00
Duration: 8 min 48 s
Description: Learn to identify neutrophil extracellular traps in feline arterial thrombi using immunofluorescence microscopy with heat-induced antigen retrieval. This protocol enables superior NET detection compared to conventional histology.

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5 minutes

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. Blunt dissect out the fascia (Figure 1B) before submerging it completely in 10% neutral-buffered formalin for a minimum of 24 h and no longer than 48 h.</li>
	<li>To dehydrate the sample, first submerge in 10% neutral-buffered formalin heated to 37 &#176;C for 1 h. Then, submerge in increasing concentrations of ethanol heated to 37 &#176;C (70%, 95%, 100%) 2x for 1 h each. Finally, without rinsing, submerge 2x in 100% toluene heated to 37 &#176;C for 1 h each.</li>
	<li>Add paraffin heated to 62 &#176;C and allow the paraffin to solidify completely overnight.</li>
	<li>Section 2&#8211;3 &#181;m of the paraffin-embedded tissue using a microtome and place on positively charged glass slides. Store sectioned tissues at -80 &#176;C until further analysis.</li>
</ol>
2. Deparaffinization, rehydration, and heat-induced antigen retrieval
<ol>
	<li>To perform deparaffinization and rehydration of sections on glass slides, place glass slides in racks and process in the following order:
	<ol>
		<li>Submerge completely in 100% xylene for 3 min. Repeat this step 2x. Do not rinse in between steps.</li>
		<li>Submerge completely in decreasing concentrations of ethanol (100%, 95%, 70%) at room temperature (RT), 3x for 3 min each. Do not rinse in between steps.</li>
		<li>Submerge completely in deionized water for 2 min. Repeat.</li>
	</ol>
	</li>
	<li>Place sections into Tris-buffered saline with 0.1 % Tween (TBST, pH = 7.6) for 2&#8211;3 min.</li>
	<li>Fill the reservoir with deionized water heated to 100 &#176;C. Allow the steamer chamber to equilibrate for 20 min. 
	NOTE: Heat-induced antigen retrieval is best performed with indirect heating generated by a steamer with a preset temperature setting, such as a food steamer.</li>
	<li>Heat the commercially available antigen retrieval solution containing Tris and EDTA (pH = 9) to 95&#8211;97 &#176;C on a temperature-controlled hot plate with constant stirring. Ensure that it does not boil. 
	NOTE: The solution should turn cloudy once it is warmed.</li>
	<li>Pour the heated antigen retrieval solution into a slide container and place the container in the chamber of the steamer. Allow the antigen retrieval solution to equilibrate to the temperature of the steamer for 3&#8211;4 min. Ensure that the temperature of the chamber is ~95 &#176;C.</li>
	<li>Submerge the slides completely in the heated antigen retrieval solution and continue the application of external heating via the steamer for 20 min.</li>
	<li>Remove the slide container from the steamer and allow the slides and the antigen retrieval solution to cool to RT. Store the diluted antigen retrieval solution at 4 &#176;C and reuse up to 2x if needed.</li>
	<li>Wash the slides 3x with TBST for 5 min.</li>
</ol>
3. Immunolabeling and autofluorescence quenching
NOTE: Table 1 details the composition of the blocking buffers used in the following steps.
<ol>
	<li>Incubate sections in Blocking Buffer 1 for 2 h at RT under gentle rocking (30&#8211;50 rpm). Seal with paraffin film to avoid drying.</li>
	<li>Without washing, immediately apply 100 &#181;L of diluted rabbit polyclonal anti-human citrullinated histone H3 (citH3) antibody (0.03 mg/mL diluted in blocking buffer 1) directly onto the slide.</li>
	<li>Place a coverslip (24 mm x 40 mm x 0.13&#8211;0.17 mm) on each section to allow even distribution of the antibody mixture.</li>
	<li>Incubate for 12&#8211;16 h at 4 &#176;C with gentle rocking (30&#8211;50 rpm). Seal with parafilm film to avoid drying.</li>
	<li>Wash 3x with TBST for 5 min.</li>
	<li>Apply 100 &#181;L of goat anti-rabbit antibody conjugated to Alexa Fluor 488 (diluted to a final concentration of 0.04 mg/mL or 1:50 in Blocking Buffer 1) as described in step 3.3. Incubate for 1 h at RT under gentle rocking (30&#8211;50 rpm). Protect slides from light.</li>
	<li>Wash with TBST 3x for 5 min.</li>
	<li>Incubate sections in Blocking Buffer 2 overnight at 4 &#176;C under gentle rocking (30&#8211;50 rpm). Protect from light.</li>
	<li>Wash with TBST 3x for 5 min.</li>
	<li>Block sections in Blocking Buffer 3 as described in step 3.3 at RT for 2 h under gentle rocking (30&#8211;50 rpm).</li>
	<li>Incubate sections with biotinylated polyclonal rabbit anti-human NE antibody (final concentration = 0.2 &#181;g/mL in Blocking Buffer 3) at 4 &#176;C for 12&#8211;16 h as described in steps 3.2&#8211;3.4.</li>
	<li>Wash with TBST 3x for 5 min.</li>
	<li>Incubate with Alexa Fluor 594 streptavidin conjugate (dilute to 1:100 or 0.02 mg/mL in Blocking Buffer 3) as described in steps 3.2&#8211;3.3 for 1 h at RT. Protect from light and seal with paraffin to prevent drying.</li>
	<li>Wash with TBST 1x for 5 min.</li>
	<li>Apply 100 &#181;L of autofluorescence quenching solution mixture directly onto the sections for 1 min as instructed by the manufacturer.</li>
	<li>Immediately wash the slides with TBST 6x for 10 min.</li>
	<li>Cover each slide with 100 &#181;L of 300 nM DAPI for 5 min in the dark.</li>
	<li>Wash with TBST for 3 min. Repeat this for a total of 5x.</li>
	<li>Apply a drop (~50 &#956;L) of antifade mounting medium, part of the autofluorescence quenching kit, directly onto the glass slide surrounding the section. Place a coverslip (24 mm x 40 mm x 0.13&#8211;0.17 mm) gently onto the section without creating any bubbles.</li>
	<li>Allow samples to cure overnight in the dark at 4 &#176;C until the mounting medium has hardened for microscopic analysis with immersion lenses.</li>
</ol>
4. Neutrophil extracellular trap identification
NOTE: The following protocol utilizes an inverted epifluorescence microscope with a 1,280 x 960 digital CCD camera (see Table of Materials).
<ol>
	<li>To locate thrombi, scan cranially to caudally along the length of the aorta, aortic bifurcation, and each femoral artery using phase contrast microscopy with a 10x objective. A thrombus is a conglomeration of tissue containing red blood cells, white blood cells, and platelets adjacent to the endothelium on phase contrast and bright field microscopy (Figure 2A, Figure 2B).</li>
	<li>First examine sections for NETs using the DAPI channel (excitation = 357/44 nm) with 10x and 20x objectives (Figure 2C). Note that cfDNA appears as decondensed DNA that is not within the confines of the cytoplasm of a cell when seen on phase contrast or bright field microscopy.</li>
	<li>Identify extracellular NE and citH3 on the Texas Red channels (excitation = 585/29 nm, emission = 628/32 nm) and green fluorescent protein channel (excitation = 470/22 nm, emission = 525/50 nm), respectively with 10, 20, and 40x objectives.</li>
	<li>Evaluate and analyze NETs within a thrombus using available software, such as Image J (NIH). NET formation is identified based on the colocalization of cfDNA, extracellular citH3, and NE as previously described18. Maintain consistent exposure time and gains of each channel throughout the acquisition of images to avoid saturation in pixel intensity.</li>
	<li>Map each thrombus based on its proximity to the descending aorta by dividing it into three equal zones, with Zone 1 closest to the aorta, Zone 3 furthest from the aorta, and Zone 2 between Zones 1 and 3). With the operator blinded to the medical condition of each subject, take at least ten random fields in each zone. Characterize the distribution of NETs in thrombi by averaging the numbers of fields with NETs in each zone or calculating the average NET-occupying area per zone.</li>
</ol>

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<li>Farkas, ÁZ., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neutrophil+extracellular+traps+in+thrombi+retrieved+during+interventional+treatment+of+ischemic+arterial+diseases%5BTitle%5D)+AND+%22Thrombosis+Research%22%5BJournal%5D)">Neutrophil extracellular traps in thrombi retrieved during interventional treatment of ischemic arterial diseases.</a> Thrombosis Research. 175, 46-52 (2019).</li>
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</ol>

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

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&amp;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&am...

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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Research

JoVE Journal

Immunology and Infection

Neutrophil extracellular traps (NETs) are networks of DNA, histones and neutrophil proteins. Although a component of the innate immune response, NETs are implicated in autoimmunity and thrombosis. This protocol describes a simple method for canine neutrophil isolation and quantification of NETs using a microplate fluorescence assay.

A Simple Fluorescence Assay for Quantification of Canine Neutrophil Extracellular Trap Release

We describe methods to isolate canine neutrophils from whole blood and visualize NET formation in live neutrophils using fluorescence microscopy. Also described are protocols to quantify NET formation and citrullinated histone H3 (citH3) expression using immunofluorescence microscopy.

In Vitro Canine Neutrophil Extracellular Trap Formation: Dynamic and Quantitative Analysis by Fluorescence Microscopy

Neutrophil extracellular traps (NETs) are three-dimensional structures generated by stimulated neutrophil granulocytes. It has become clear in recent years that NETs are involved in a wide variety of diseases. Detection of NETs in tissue may have diagnostic relevance, so standardized protocols for labelling NET components are required.

Immunofluorescence Labelling of Human and Murine Neutrophil Extracellular Traps in Paraffin-Embedded Tissue

Neutrophil extracellular traps (NETs) are associated with various diseases, and immunofluorescence is often used for their visualization. However, there are various staining protocols, and, in many cases, only one type of tissue is examined. Here, we establish a generally applicable protocol for staining NETs in mouse and human tissue.

Immunofluorescence Imaging of Neutrophil Extracellular Traps in Human and Mouse Tissues

Neutrophil Extracellular Traps (NETs) are an important innate immune mechanism to fight pathogenic bacteria, fungi and parasites. Here we describe methods to isolate neutrophil granulocytes from human blood and to activate them to form NETs. We present preparation techniques to visualize NETs in light and electron microscopy.

Neutrophil Extracellular Traps: How to Generate and Visualize Them

In the following protocol, we describe a very simple way to isolate Neutrophil Extracellular traps (NETs) from human whole blood using readily available reagents. We then demonstrate how the isolated NETs can be used in an in vitro adhesion assay with cancer cells.

Simplified Human Neutrophil Extracellular Traps (NETs) Isolation and Handling

Here a method to localize bacteria within paraffin-embedded tissues using DIG-labeled 16S rRNA-targeting DNA probes has been described. This protocol can be applied to study the role of bacteria in various diseases such as periodontitis, cancers, and inflammatory immune diseases.

In Situ Detection of Bacteria within Paraffin-embedded Tissues Using a Digoxin-labeled DNA Probe Targeting 16S rRNA

Lipids are known to play an important role in cellular functions. Here, we describe a method to determine the lipid composition of neutrophils, with emphasis on the cholesterol level, by using both HPTLC and HPLC to gain a better understanding of the underlying mechanisms of neutrophil extracellular trap formation.

Methods to Study Lipid Alterations in Neutrophils and the Subsequent Formation of Neutrophil Extracellular Traps

We describe here a method for obtaining and identifying a newly characterized subpopulation of neutrophil-derived giant phagocytes. These cells develop in culture from fresh human blood neutrophils, and are characterized by phagocytosis, autophagy, immensely large size, and extended lifespan. This method is essential to further investigate this unique neutrophil-derived subpopulation.

Development and Identification of a Novel Subpopulation of Human Neutrophil-derived Giant Phagocytes In Vitro

Macrophage extracellular traps are a newly described entity. This article will concentrate on confocal microscopy methods and how they are visualized in vitro and in vivo from lung samples.

Visualizing Macrophage Extracellular Traps Using Confocal Microscopy

Learn to identify neutrophil extracellular traps in feline arterial thrombi using immunofluorescence microscopy with heat-induced antigen retrieval. This protocol enables superior NET detection compared to conventional histology.

Identification of Neutrophil Extracellular Traps in Paraffin-Embedded Feline Arterial Thrombi using Immunofluorescence Microscopy (Video) | JoVE Journal

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

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