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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.
The mRNA in situ hybridization technique is a useful tool that allows the specific and selective labeling of RNA sequences in brain slices in a cell-dependent manner (Grabinski et al., 2015). Furthermore, the use of antibodies against these specific cytokines can produce variable results due to the detection limits of the technique. Namely, because these cytokines are expressed in low abundance, the detection limit becomes the limiting factor for the use of antibodies. Lastly, fluorescence in situ hybridization (FISH) combined with immunohistochemistry (ISH) allows the examination of cytokine mRNA profiles in distinct cells with high selectivity and specificity, thus allowing us to identify the precise cellular source of cytokine production following TBI. This protocol describes how the combination of FISH and immunofluorescence imaging can bridge the gap between mRNA and protein analysis. We can identify the target mRNA being produced by microglia/macrophage cells. Analysis of both RNA and protein expression in the same tissue allows differentiating between cell specific production of microglia/macrophage cells and other cell types. There are limited studies on the effects of sex on inflammation profile following traumatic brain injury (TBI). In our recent publication (Villapol et al., 2017), we used RNAscope® technology combined with immunofluorescence to determine pro-inflammatory (e.g., IL1β and TNFα) and anti-inflammatory (e.g., TGFβ and Arg1) cytokine mRNA expression profiles in microglia/macrophages in the injured brains of male and female mice. Our data demonstrate that a mixed pattern of both pro-inflammatory and anti-inflammatory cytokine expression occurred in microglia/macrophages in the first week after TBI. Also, we have previously shown that IL-10 levels are significantly increased in microglia/macrophages in the injured cortex of NOX2−/− mice (Barrett et al., 2017).
In summary, the use of FISH improves specificity and sensitivity when examining cytokine mRNAs in distinct cells, confirmed by antibody co-immunostaining for microglia/macrophages. This method allows us to identify the cellular source of cytokine production following brain injury with much-improved confidence.
Fluorescent in situ hybridization was performed using RNAscope® Technology 2.5 Red fluorescent kit for fresh frozen tissue, with some modifications.
All target probes consist of 20 short double-Z oligonucleotide probe pairs that are gene specific and were obtained from ACD.
We show the critical steps for the FISH technique described in this protocol as follows: boil the slides with the antigen retrieval solution, place slides on ACD EZ-slide holder/rack into the HybEZ™ Oven, create a barrier around tissue sections, add 60–100 µl per probe or amplification solution to each section, move the slide rack up and down, and acquire representative confocal images.
This work was supported by NIH grants R03NS095038 (S. Villapol), R01NS067417 (M.P. Burns), and the Dean of Biomedical Research (Toulmin pilot project) provided by the Georgetown University Medical Center (I. Mocchetti). We would like to say thanks to Dr. Kathy Maguire-Zeiss for letting us use her fluorescence microscope, and to Lucas Djavaherian for his help recording the video. This protocol has been adapted from (Barrett et al., 2017, Villapol et al., 2017).
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.