PMID- 32281975
OWN - NLM
STAT- MEDLINE
DCOM- 20200909
LR  - 20200909
IS  - 1940-087X (Electronic)
IS  - 1940-087X (Linking)
IP  - 157
DP  - 2020 Mar 29
TI  - Identification of Neutrophil Extracellular Traps in Paraffin-Embedded Feline 
      Arterial Thrombi using Immunofluorescence Microscopy.
LID - 10.3791/60834 [doi]
AB  - 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.
FAU - Duler, Laetitia
AU  - Duler L
AD  - Veterinary Medical Teaching Hospital, School of Veterinary Medicine, University 
      of California, Davis.
FAU - Nguyen, Nghi
AU  - Nguyen N
AD  - Department of Veterinary Surgical and Radiological Sciences, School of Veterinary 
      Medicine, University of California, Davis.
FAU - Ontiveros, Eric
AU  - Ontiveros E
AD  - Department of Veterinary Medicine and Epidemiology, School of Veterinary 
      Medicine, University of California, Davis.
FAU - Li, Ronald H L
AU  - Li RHL
AD  - Department of Veterinary Surgical and Radiological Sciences, School of Veterinary 
      Medicine, University of California, Davis; rhli@ucdavis.edu.
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PT  - Video-Audio Media
DEP - 20200329
PL  - United States
TA  - J Vis Exp
JT  - Journal of visualized experiments : JoVE
JID - 101313252
SB  - IM
MH  - Animals
MH  - Arteries/*metabolism
MH  - Cats
MH  - Extracellular Traps/*metabolism
MH  - Microscopy, Fluorescence/*methods
MH  - Neutrophils/*metabolism
MH  - Paraffin Embedding/*methods
EDAT- 2020/04/14 06:00
MHDA- 2020/09/10 06:00
CRDT- 2020/04/14 06:00
PHST- 2020/04/14 06:00 [entrez]
PHST- 2020/04/14 06:00 [pubmed]
PHST- 2020/09/10 06:00 [medline]
AID - 10.3791/60834 [doi]
PST - epublish
SO  - J Vis Exp. 2020 Mar 29;(157). doi: 10.3791/60834.