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

Summary

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.

Abstract

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.

Introduction

Cats with hypertrophic cardiomyopathy are at risk of life-threatening thromboembolic complications^1,2. 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 ar....

Protocol

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’ consent.

1. Tissue fixation, embedding, and sectioning

1. 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 minimu....

Results

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&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.......

Discussion

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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