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 …
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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 |
