Characterization of Macrophage Extracellular Traps in Mouse Lung Tissue Samples

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).