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Bio Protoc. 2017 Nov 20;7(22). pii: e2608. doi: 10.21769/BioProtoc.2608.

Combination of Fluorescent in situ Hybridization (FISH) and Immunofluorescence Imaging for Detection of Cytokine Expression in Microglia/Macrophage Cells.

Author information

1
Department of Neuroscience, Georgetown University, Washington, DC, United States.
2
Department of Anesthesiology, Center for Shock, Trauma, and Anesthesiology Research (STAR), University of Maryland School of Medicine, Baltimore, MD, United States.

Abstract

Microglia and macrophage cells are the primary producers of cytokines in response to neuroinflammatory processes. But these cytokines are also produced by other glial cells, endothelial cells, and neurons. It is essential to identify the cells that produce these cytokines to target their different levels of activation. We used dual RNAscope® fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) techniques to visualize the mRNA expression pattern of pro- and anti-inflammatory cytokines in microglia/macrophages cells. Using these methods, we can associate one mRNA to specific cell types when combining with different cellular markers by immunofluorescence. Results from RNAscope® probes IL-1β, TNFα, TGFβ, IL-10 or Arg1, showed colocalization with antibodies for microglia/macrophage cells. These target probes showed adequate sensitivity and specificity to detect mRNA expression. New FISH detection techniques combined with immunohistochemical techniques will help to jointly determine the protein and mRNA localization, as well as provide reliable quantification of the mRNA expression levels.

KEYWORDS:

Cytokines; Immunofluorescence; Macrophages; Microglia cells; Neuroinflammation; RNAscope; Traumatic brain injury (TBI); in situ hybridization

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