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

荧光原位杂交（FISH）与免疫荧光成像联合检测小胶质细胞/巨噬细胞中细胞因子的表达

Maria Fe Lanfranco Maria Fe Lanfranco
DL David J. Loane
IM Italo Mocchetti
MB Mark P. Burns
Sonia Villapol Sonia Villapol

发布: 2017年11月20日第7卷第22期 DOI: 10.21769/BioProtoc.2608 浏览次数: 15892

评审: Xi FengKarthik KrishnamurthyAnonymous reviewer(s)

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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: in situ hybridization (原位杂交) search RNAscope (RNAscope) search Traumatic brain injury (TBI) (创伤性脑损伤（TBI）) search Macrophages (巨噬细胞) search Immunofluorescence (免疫荧光) search Microglia cells (小胶质细胞) search Neuroinflammation (神经炎症) search Cytokines (细胞因子) search

Background

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.

Materials and Reagents

  1. Thick Whatman paper (Fischerbrand® Chromatography Paper) (Fisher Scientific, catalog number: 05-714-4 )
  2. Gelatin-coated glass slides (Superfrost Plus) (Fisher Scientific, catalog number: 12-550-15 )
  3. Microscope cover glass (24 x 50 mm) (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 3422ERI )
  4. ImmEdge hydrophobic barrier pen (Advanced Cell Diagnostics, catalog number: 310018 )
  5. Kimwipes (KCWW, Kimberly-Clark, catalog number: 34133 )
  6. Foil paper
  7. Mice (C57BL/6 from THE JACKSON LABORATORY, catalog number: 000664 )
  8. RNAscope® positive control probe-Mm-Ppib (peptidylprolyl isomerase B) (Advanced Cell Diagnostics, catalog number: 313911 )
  9. RNAscope® negative control probe-DapB (Advanced Cell Diagnostics, catalog number: 310043 )
  10. RNAscope® probe-Mm-Tgfβ1 (Transforming growth factor beta 1) (Advanced Cell Diagnostics, catalog number: 407751 )
  11. RNAscope® probe-Mm-IL1β (interleukin 1 beta) (Advanced Cell Diagnostics, catalog number: 316891 )
  12. RNAscope® probe-Mm-TNFα (tumor necrosis factor alpha) (Advanced Cell Diagnostics, catalog number: 311081 )
  13. RNAscope® probe-Mm-Arg1 (Arginase 1) (Advanced Cell Diagnostics, catalog number: 403431 )
  14. Phosphate-buffered saline (PBS), 10x solution (Fisher Scientific, catalog number: BP39920 )
  15. Paraformaldehyde (PFA) stock to prepare 4% (w/v) PFA, PRILLS (Electron Microscopy Sciences, catalog number: 19200 )
  16. Sucrose Crystalline stock (Fisher Scientific, catalog number: S5 ) to prepare 30% (w/v) solution diluted in ultra-pure water
  17. Ultra-pure water (e.g., Milli-Q)
  18. Freshly prepared 50% (v/v) ethanol in ultra-pure water (e.g., Milli-Q water) solution
  19. Freshly prepared 70% (v/v) ethanol in ultra-pure water (e.g., Milli-Q water) solution
  20. Absolute ethanol (200-proof ethanol) (Fisher Scientific, catalog number: BP28184 )
  21. RNAscope® H2O2 & protease plus reagents (Advanced Cell Diagnostics, catalog number: 322330 )
  22. RNAscope® 2.5 HD detection reagents-RED (Advanced Cell Diagnostics, catalog number: 322360 ) (read Note 1)
  23. RNAscope® target retrieval reagents (Advanced Cell Diagnostics, catalog number: 322000 )–previously known as pretreatment 2 solution
  24. Normal goat serum (NGS) blocking solution (Vector Laboratories, catalog number: S-1000 )
  25. Triton X-100
  26. Polyclonal anti-rabbit Iba-1 (ionized calcium binding adaptor molecule-1) antibody (Wako Pure Chemical Industries, catalog number: 019-19741 )
  27. Polyclonal anti-rabbit P2Y12 antibody (AnaSpec, catalog number: AS-55042A )
  28. Polyclonal anti-rat F4/80 antibody (R&D systems, catalog number: MAB5580 )
  29. Goat anti-rabbit Alexa Fluor® 488 secondary antibody (Thermo Fisher Scientific, catalog number: A-11034 )
  30. Goat anti-rat Alexa Fluor® 488 secondary antibody (Thermo Fisher Scientific, catalog number: A-11006 )
  31. Fluoro-gel (with Tris buffer) mounting medium, 20 ml (Electron Microscopy Science, catalog number: 17985-10 )
  32. Glycerol
  33. Ethylene glycol
  34. RNAscope® Wash Buffer Reagents (4 x 60 ml) (Advanced Cell Diagnostics, catalog number: 310091 )
  35. 4’,6-Diamidino-2-phenylindole (DAPI) solution (Sigma-Aldrich, catalog number: D9542 )
  36. Antifreeze solution (see Recipes)
  37. 1x wash buffer (see Recipes)
  38. 1x antigen retrieval solution (see Recipes)
  39. DAPI solution (see Recipes)

Equipment

  1. GilsonTM PIPETMAN ClassicTM Pipets (Gilson, models: P20, P200, P1000, catalog numbers: F123600 , F123601 , F123602 )
  2. 2 L beaker
  3. Extra-long forceps (Thermo Fisher Scientific, FisherbrandTM, catalog number: 10-316A )
  4. Timer, TraceableTM NanoTM (Fisher Scientific, FisherbrandTM, catalog number: 14-649-83 )
  5. Slide Holder Handle, 24 slides (Electron Microscopy Sciences, catalog number: 62543-06 )
  6. Sliding microtome, MicromHM 430 (Thermo Fisher Scientific, Thermo ScientificTM, model: HM 430 , catalog number: 910010)
  7. HybEzTM hybridization system (Advanced Cell Diagnostics, catalog number: 310010 )
  8. ACD HybEZTM humidity control tray with lid (Advanced Cell Diagnostics, catalog number: 310012 )
  9. Tissue Tek Slide Stain Set with EasyDipTM slide staining (Electron Microscopy Sciences, catalog number: 62540-01 )
  10. ACD EZ-slide holder/rack (Advanced Cell Diagnostics, catalog number: 310017 )
  11. Thermix® Hot plate with magnetic stirrer, Model 210T (Fisher Scientific, model: Model 210T , catalog number: 11-493-210T)
  12. Fluorescent images were acquired on an Axioplan 2 microscope (Carl Zeiss, model: Axioplan 2 , catalog number: 451485) with a Photometrics camera (CoolSNAP, fx, Axioskop, Roper Scientific, serial number: A02M86017)
  13. Leica SP8 confocal microscope (Leica Microsystems, model: Leica TCS SP8 )

Software

  1. GraphPad Prism software V. 5.0 (GraphPad Software, Inc., La Jolla, CA)
  2. Adobe Photoshop CS5 V. 12.0 (Adobe Photoshop, CC)
  3. AxionVision 4V, 4.8.2.0., licensed to: 3018897 (Carl Zeiss MicroImaging, GmbH)
  4. ImageJ64 software (National Institute of Health)

Procedure

English
中文翻译

文章信息

版权信息

© 2017 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Lanfranco, M. F., Loane, D. J., Mocchetti, I., Burns, M. P. and Villapol, S. (2017). Combination of Fluorescent in situ Hybridization (FISH) and Immunofluorescence Imaging for Detection of Cytokine Expression in Microglia/Macrophage Cells. Bio-protocol 7(22): e2608. DOI: 10.21769/BioProtoc.2608.

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分类

神经科学 > 神经系统疾病 > 动物模型

神经科学 > 细胞机理 > 胞内信号传导

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