Characterization of Macrophage Extracellular Traps in Mouse Lung Tissue Samples

Published: August 31, 2023

Abstract

Source: Sharma, R., et al. Visualizing Macrophage Extracellular Traps Using Confocal Microscopy. J. Vis. Exp. (2017)

In this video, we describe a protocol for the immunostaining and confocal microscopy-based visualization of macrophage extracellular traps (METs) in murine lung tissue. Tissue-resident macrophages are characterized by the expression of the macrophage-specific F4/80 surface glycoprotein marker, while METs are visualized by labeling the MET markers matrix metalloproteinase 9, citrullinated histone H3, and extracellular chromatin.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board. 1. Lung Tissue Samples NOTE: For in vivo studies of lung tissue, study the samples after the relevant exposure. The exposure that is most relevant for this experiment is infectious microorganisms, particularly bacteria. Mouse strains that could be used for this method are C57BL/6 or …

開示

The authors have nothing to disclose.

Materials

Mouse samples: Primary antibodies
Goat anti-mouse MMP9 Abcam AF909 10 ug/ml
Rabbit anti-mouse H3Cit (Citrulline R26) Abcam AB5103 10 ug/ml
Rat anti-mouse F4/80 In-house (hybridoma) In house 20 ug/ml
Sheep anti-human Anti-HNE /NE Sapphire Bioscience LS-B4244 25 ug/ml
Mouse anti-human PADI4/PAD4 Abcam AB128086 10.1 ug/ml
Super-frost plus slides Menzel S41104A
Dapi-prolong gold Molecular probes P36931
Secondary antibodies
Chicken anti-rabbit AF 488/ Life technologies A-21441
Chicken anti-rabbit AF 594 Life technologies A-21442
Chicken anti-goat AF 594 Life technologies a-21468
Chicken anti-mouse AF488 Life technologies A-21200
Donkey anti-sheep AF 594 Life technologies A-11018
Chicken anti-mouse AF 647 Life technologies A-21463
Donkey anti-sheep AF 594 Life technologies A-11016
Isotype control
Rabbit IgG In house
Mouse IgG1  BD Bioscience 550878
Mouse IgG2a  BioLegend  400201
Sheep IgG  In house
Microscopes
C1 Confocal scanning microscope Nikon
FV1200 Confocal scanning microscope Olympus
Tissue sources
Mouse lung tissue from the Department of Pharmacology, University of Melbourne.
Media
RPMI Sigma-Aldrich r8758
Fetal calf serum Sigma-Aldrich F0926
L-glutamine Sigma-Aldrich G7513
Other reagents
Paraformaldehyde/periodate/lysine (PLP) fixative Sigma-Aldrich 27387
Xylene Sigma-Aldrich 214736
Natural formalin Sigma-Aldrich 42904
Paraffin Sigma-Aldrich 327204
Ketamine  Sigma-Aldrich  K2753
Agarose  Sigma-Aldrich  A2576
Solvent 3B  Hi-Chem  2026
Coverslips 12 ml  Sigma-Aldrich  S1815
Coverslips 60 x 24 ml  Sigma-Aldrich  C6875
Mice
c57 black 6 Monash animal research platform (MARP)
BALB/c MARP

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記事を引用
Characterization of Macrophage Extracellular Traps in Mouse Lung Tissue Samples. J. Vis. Exp. (Pending Publication), e21567, doi: (2023).

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