Immunofluorescence Staining Using IBA1 and TMEM119 for Microglial Density, Morphology and Peripheral Myeloid Cell Infiltration Analysis in Mouse Brain
Abstract
This is a protocol for the dual visualization of microglia and infiltrating macrophages in mouse brain tissue. TMEM119 (which labels microglia selectively), when combined with IBA1 (which provides an exceptional visualization of their morphology), allows investigation of changes in density, distribution, and morphology. Quantifying these parameters is important in providing insights into the roles exerted by microglia, the resident macrophages of the brain. Under normal physiological conditions, microglia are regularly distributed in a mosaic-like pattern and present a small soma with ramified processes. Nevertheless, as a response to environmental factors (i.e., trauma, infection, disease, or injury), microglial density, distribution, and morphology are altered in various manners, depending on the insult. Additionally, the described double-staining method allows visualization of infiltrating macrophages in the brain based on their expression of IBA1 and without colocalization with TMEM119. This approach thus allows discrimination between microglia and infiltrating macrophages, which is required to provide functional insights into their distinct involvement in brain homeostasis across various contexts of health and disease. This protocol integrates the latest findings in neuroimmunology that pertain to the identification of selective markers. It also serves as a useful tool for both experienced neuroimmunologists and researchers seeking to integrate neuroimmunology into proje…
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Images were blinded to experimental groups for density and morphology analyses.
“The images were blinded to the experimental groups and analysed using Fiji (NIH; v.1.53), as previously reported [56, 64].”
From Sex chromosomes and sex hormones differently shape microglial properties during normal physiological conditions in the adult mouse hippocampus (2025) · cites this paper as [64]
Ultrastructural analyses were performed using QuPath on images blinded to both animal and group.
“The microglial ultrastructural analyses was performed using QuPath (v.0.4.4) [71] on images blinded to both animal and group, following an adaptation from our previous work [56, 64].”
From Sex chromosomes and sex hormones differently shape microglial properties during normal physiological conditions in the adult mouse hippocampus (2025) · cites this paper as [64]
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- Authors
- González Ibanez, F., Picard, K., Bordeleau, M., Sharma, K., Bisht, K., & Tremblay, M.-È.
- Affiliations
- Université Laval (CA)
- Centre de recherche du CHU de Québec-Université Laval (CA)
- Venue
- Journal of Visualized Experiments
- Publisher
- MyJove Corporation
- Published
- 2019-10-27
- Issue
- 152
- ISSN
- 1940-087X (ISSN-L)
- OpenAlex
- W2981952575
- Cited by
- 77
- References
- 22
- Citations by year
- 2026: 10 · 2025: 11 · 2024: 12 · 2023: 14 · 2022: 12 · 2021: 10 · 2020: 8
- FWCI
- 3.30
- Type
- journal article
- Language
- en
- Open access
- closed
- Locations
- Journal of Visualized Experiments (publishedVersion)
- PubMed (publishedVersion)
- Topics
- Life Sciences › Neuroscience › Neurology › Neuroinflammation and Neurodegeneration Mechanisms
- Life Sciences › Neuroscience › Cellular and Molecular Neuroscience › Neuroscience and Neuropharmacology Research
- Life Sciences › Neuroscience › Neurology › Neurological Disease Mechanisms and Treatments
- Keywords
- microglia, TMEM119, Iba1, infiltrating macrophages, microglial density, microglial morphology, mouse brain, neuroimmunology, brain homeostasis, microglial activation
- MeSH terms
- Animals, Brain, Fluorescent Antibody Technique, Homeostasis, Macrophages, Staining and Labeling, Microglia, Myeloid Cells, Mice
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