Characterization of Macrophage Extracellular Traps in Mouse Lung Tissue Samples

Overview

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 BALB/c mice, 10 - 12 weeks old, weight 25 - 30 g, and male.

1. Euthanize mice, via an intraperitoneal injection of ketamine (400 mg/kg) and xylazine (40 mg/kg). Open the thoracic cavity and expose the lungs and heart using surgical forceps and scissors.
   1. Infuse warm PBS using a 21-gauge needle into the right ventricle to wash out the blood from the vessels for 30 s, then infuse 10 mL of 2% PLP fixative for 30 s (into the right ventricle).
   2. Use warm PBS (42 °C) to lavage the open chest cavity. Intubate the trachea with a 21-gauge needle and a piece of 0.8 mm tubing cut to size, and inject 800 μL of pre-warmed (to 42 °C), low-melting point 3% agarose solution.
   3. Tie off the trachea with braided silk (4.0 USP). Place the thread around the trachea, just below the insertion point of the cannula, and secure it via a simple overhand knot. To solidify the agarose, administer ice-cold PBS around the lungs. Remove the lungs and heart as a block from the thoracic cavity using both scissors and blunt dissection. Remove each lung individually and then fix them for 16 h in 2% PLP or formalin at 4 °C.
   4. Store the specimens in PBS at 4 °C until tissue sectioning.
2. To prepare the lung tissue samples use the following steps.
   1. Fix lung tissues (resected in step 1.1) in 10% natural-buffered formalin for 24 h at room temperature. Transfer to 75% ethanol and put in a tissue processor on a 12 h cycle, (2.5 h in 75% EtOH, 3 h in absolute EtOH, 3.5 h in solvent 3B, and 3 h in paraffin).
   2. Embed in standard paraffin to create formalin-fixed, paraffin-embedded (FFPE) blocks which are stored at room temperature.
      NOTE: At this stage, it is generally convenient to cut the lung sections for standard slides.
   3. Cut the FFPE lung sections at 4 - 5 μm thickness using a microtome and mount them on charged, coated slides.
   4. To mount slides, put 3 drops of mounting medium on 60 mm x 24 mm coverslips (size 1.5), invert the slide on the coverslip, and push out excess medium. Hermetically seal them with nail polish and store them at room temperature.

2. Preparation/Microscopy of Lung Tissue Samples

1. To de-wax, rehydrate and pretreat the FFPE lung tissue sample with antigen retrieval solution.
   1. Oven dry the FFPE slides for 60 min at 60 °C. Then put the slides in xylene solution for 30 min and transfer to 70% ethanol solution for 5 min at room temperature. Rinse in tap water.
   2. Place in heat-proof plastic wrap and subject to HIER-antigen retrieval in a pressure cooker for 10 min in 10 m/mol/L Tris, 1 mmol/EDTA pH 9.0. Cool for 20 min. Wash twice in tap water for 5 min on a rocker, then once with PBS on a rocker.
   3. Block with 10% chicken serum in 5% BSA/PBS for 30 min at room temperature.
2. Stain the tissue.
   1. To define METs in macrophages, add primary antibodies (MMP-9, H3Cit, and F4/80) in 1% BSA/PBS for 16 h at 4 °C at 1/100 dilution (specific details about antibody amounts are listed in Table of Materials). Wash two times with PBS for 5 min on a rocker.
   2. Achieve fluorescent detection by incubation with corresponding secondary antibodies in 1% BSA/PBS for 40 min at room temperature. Antibodies are: (1) chicken anti-mouse 488 (green), (2) chicken anti-goat 594 (red), and (3) donkey anti-mouse 647 (far red).
   3. Wash the sections in PBS and mount them with a DAPI-containing mounting medium for chromatin staining.
3. Obtain fluorescent images using a confocal laser scanning head attached to an inverted microscope.
   1. Excite the preparation with 405, 488, 561, and 647 nm lasers. Capture single plane 512 x 512-pixel images by clicking on the line-sequential, leveling button (2 line averaging) using 20X 0.1 NA air and 40X 1.0 NA oil objectives.
   2. Obtain at least ten fields of view per section for analysis and data for each result (i.e., 10 high-power fields of view (HFOV) for the control, 10 for the stimulated sample, etc., for each mouse).
      NOTE: To obtain a representative sample of the whole field, a matrix system can be used in which the field is divided into different sections, and for each different sample the same matrix is used to select fields of view (e.g., the field can be divided into 9 sections, and from the center and the 4 corners, two HFOV are taken).

Materials

Name	Company	Catalog Number	Comments
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