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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   

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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), RNAscope(RNAscope), Traumatic brain injury (TBI)(创伤性脑损伤(TBI)), Macrophages(巨噬细胞), Immunofluorescence(免疫荧光), Microglia cells(小胶质细胞), Neuroinflammation(神经炎症), Cytokines(细胞因子)

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

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.

  1. RNAscope® Probe-Mm-Arg1 (accession number NM_007482.3, target region 2-1114)
  2. RNAscope® Probe-Mm-TNFα (accession number NM_013693.2, target region 41-1587)
  3. RNAscope® Probe-Mm-Tgfβ1 (accession number NM_011577.1, target region 588-1913)
  4. RNAscope® Probe-Mm-IL1β (accession number NM_008361.3, target region 2-950)
  5. RNAscope® Positive Control Probe-Mm-Ppib (accession number NM_011149.2, target region 98-856)
  6. RNAscope® Negative Control Probe-DapB (targets a bacterial gene)

  1. Tissue
    1. Euthanize mice with CO2 and transcardially perfuse with ice-cold phosphate buffered saline (PBS) as was previously described (Villapol et al., 2017).
    2. Post fix the whole brains with 4% (v/v) PFA overnight.
    3. Transfer the mice brains into a 30% (v/v) sucrose solution in PBS for dehydration for 48 h at 4 °C, or until the brain sinks completely to the bottom of the tube.
    4. Section the mice brains at 20 μm-thick in coronal orientation with a sliding microtome.
    5. Cryoprotect brains in an antifreeze solution (see Recipes) and store at -20 °C.

  2. Before starting
    1. Prepare 1.5 L of 1x wash buffer (see Recipes).
    2. Set HybEZTM hybridization system to 40 °C.
    3. Cut Whatman paper and place at center on the bottom of the HybEZTM humidity tray and add approximately 50 ml of ultra-pure water.
    4. Warm HybEZTM humidity control tray containing wet Whatman paper for 30 min before use.
    5. Allow ‘Pretreat 1’ solution (hydrogen peroxide solution) to equilibrate at room temperature (RT) before use.
    6. Equilibrate amplification reagents (AMP solution 1-6, obtained from ACD) at RT.
    7. Warm controls and target probes for 10 min at 40 °C and cool at RT before use.

  3. RNAscope® Technology for Fluorescent in situ hybridization (FISH)
    1. Mount coronal brain sections (20 μm-thick) on gelatin-coated slides and store at -80 °C until use. Attempt to locate brain sections on the bottom of the slide to avoid using an excessive amount of buffer during washes.
    2. Place slides on a vertical plastic staining rack and immerse in 50% freshly made ethanol solution for 5 min.
    3. Transfer the vertical plastic staining rack containing the slides into an EasyDipTM slide staining system containing 70% ethanol solution for 5 min (Figure 1A and Video 1).
    4. Transfer the vertical plastic staining rack containing the slides into an EasyDipTM slide staining system containing 100% ethanol solution for 5 min.
    5. Transfer the vertical plastic staining rack containing the slides into an EasyDipTM slide staining system containing a second 100% ethanol solution for 5 min (read Note 1).
    6. Remove slides from 100% EtOH, and let them air dry completely at RT for 30 min in a slide holder.
    7. Draw a hydrophobic barrier around each section using an immEdge hydrophobic barrier pen (Figure 1B and Video 1).
    8. From this step forward, do not allow the brain sections to dry out completely.
    9. Pretreatment 1 (hydrogen peroxide treatment)
      1. Place slides on ACD EZ-slide holder/rack and add 60-100 μl per brain section of pretreat solution 1 to each section (Figure 1C and Video 1, read Note 2).
      2. Incubate the brain section in pretreat solution 1 for 10 min at RT. Start the timer once you add the pretreat solution 1 to the first slide.
      3. Decant the pretreat solution 1 by flicking the slides into a waste container or by tapping the side of the slides on a Kimwipe if necessary.
      4. Place slides into the vertical plastic staining rack submerged in ultra-pure water.
      5. Wash slides by moving the staining rack up and down several times.
    10. Pretreatment 2 (antigen retrieval solution)
      1. Bring 350 ml 1x antigen retrieval solution (pretreatment solution 2) (see Recipes) to a boil using a hot plate. (It usually takes 40 min to reach boiling.) It’s recommendable to cover the container with foil paper.
      2. Check to ensure the temperature is between 100-104 °C.
      3. Slowly submerge the vertical plastic staining rack with slides using long curved forceps into the boiling antigen retrieval solution.
      4. Check to ensure the temperature is between 100-104 °C.
      5. Boil slides for 2 to10 min (it depends on the quality of tissue).
      6. Rinse slides in ultra-pure water (repeat once more).
    11. Pretreatment 3 (protease digestion)
      1. Decant the ultra-pure water by flicking the slides into a waste container or by tapping the side of the slides on a Kimwipe if necessary.
      2. Place slides on the ACD EZ-slide holder/rack.
      3. Add about 60-100 μl per brain section of pretreatment solution 3 (protease solution) to cover entire tissue section (Figure 1D and Video 1, read Note 2).
      4. Place the ACD EZ-slide holder/rack into the pre-warmed HybEZTM Oven with lid and incubate for 30 min at 40 °C.
      5. Remove the ACD EZ-slide holder/rack, and place the ACD HybEZTM humidity control tray back into the HybEZTM Oven hybridization system (Figure 1E and Video 1).


        Figure 1. Components of the RNAscope protocol. A. Washes in distilled water; B. Draw a hydrophobic barrier around each section; C. Add pretreatment solutions; D. Amplification solutions; E. Holder/rack.

        Video 1. Critical steps of the Fluorescent in situ hybridization (FISH) protocol. 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 HybEZTM 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.

      6. Decant the pretreatment solution 3 by flicking the slides into a waste container or by tapping the side of the slides on a Kimwipe if necessary.
      7. Place slides into the vertical plastic staining rack submerged in ultra-pure water.
      8. Wash slides by moving the slide rack up and down several times.
    12. Target probe hybridization
      1. Decant the ultra-pure water by flicking the slides into a waste container or by tapping the side of the slides on a Kimwipe if necessary.
      2. Place slides on the ACD EZ-slide holder/rack (read Note 3).
      3. Add 60-100 μl per brain section of target probes or control probes to cover entire tissue section (read Note 2).
      4. Place the ACD EZ-slide holder/rack into the pre-warmed ACD HybEZTM humidity control tray and incubate for 2 h at 40 °C in the HybEZTM hybridization system.
      5. Remove the ACD EZ-slide holder/rack, and place the ACD HybEZTM humidity control tray back into the HybEZTM hybridization system.
      6. Decant the control and target probes by flicking the slides into a waste container or by tapping the side of the slides on a Kimwipe.
      7. Place slides into the vertical plastic staining rack submerged in 1x wash buffer.
      8. Wash slides by moving the slide rack up and down several times for 2 min.
    13. Amplification steps (AMP 1-AMP 6)
      1. Decant the 1x wash buffer by flicking the slides into a waste container or by tapping the side of the slides on a Kimwipe.
      2. Place slides on ACD EZ-slide holder/rack, do not let the tissue sections dry out completely.
      3. Add 60-100 μl per brain section of AMP solution to each tissue section (read Note 2).
      4. Place the ACD EZ-slide holder/rack into the ACD HybEZTM humidity control tray and incubate according to Table 1.

        Table 1. Amplification solutions and their corresponding incubation times and temperatures


      5. Decant AMP solution by flicking the slides into a waste container or by tapping the side of the slides on a Kimwipe.
      6. Place slides on a vertical plastic staining rack submerged into 1x wash buffer.
      7. Wash slides by moving the slide rack up and down several times for 2 min.
    14. Detect the signal (10 min at RT)
      1. Mix 60 μl of Red-A and 1 μl of Red-B solutions per slide. Protect from light.
      2. Decant the 1x wash buffer by flicking the slides into a waste container or by tapping the side of the slides on a Kimwipe.
      3. Place slides on ACD EZ-slide holder/rack, do not let the tissue sections dry out completely.
      4. Add 60 μl of detection solution to each tissue section (read Note 2). Make sure to start with the positive and negative control slides.
      5. Incubate for 10 min at RT inside the ACD HybEZTM humidity control tray with lid. Protect from light. Start the timer immediately after adding the detection solution to the first slide.
      6. Check every two minutes the progression of the hybridization signal by comparing the positive and negative controls. The positive control should be reddish in color, whereas the negative control should show little to no background.
      7. Decant the detection solution by flicking the slides into a waste container or by tapping the side of the slides on a Kimwipe.
      8. Place slides on a vertical plastic staining rack submerged into ultra-pure water.
      9. Wash slides by moving the slide rack up and down several times for 2 min.
      10. Before proceeding with immunohistochemistry, check the hybridization signal under the microscope. Positive hybridization signal consists of several punctate signals (red dots) representing the mRNA transcript (Wang et al., 2012) (Figure 2).


        Figure 2. Representative images of positive control probes and nuclei (DAPI, blue) on selected brain sections. Scale bar = 50 μm.

    15. Immunofluorescence
      1. Transfer the vertical plastic staining rack containing the slides into an EasyDipTM slide staining system containing PBS.
      2. Wash slides by moving the slide rack up and down several times. Repeat three times.
      3. Block in 5% (v/v) normal goat serum (NGS) in PBS supplemented with 0.3% (v/v) Triton X-100 (PBST) for 1 h at RT (or overnight at 4 °C) inside the ACD HybEZTM humidity control tray with lid.
      4. Incubate in primary antibody in 5% (v/v) NGS in PBST overnight at 4 °C at the following dilutions:
        polyclonal anti-rabbit Iba-1 (1:500)
        polyclonal anti-rabbit P2Y12 (1:1,000)
        polyclonal anti-rat F4/80 (1:200)
      5. Place slides on a vertical plastic staining rack submerged into PBS. Wash slides by moving the slide rack up and down several times. Repeat three times for 5 min each.
      6. Incubate with corresponding Alexa Fluor® 488 secondary antibodies (1:1,000 in 5% (v/v) NGS in PBST) for 1-2 h at RT inside the ACD HybEZTM humidity control tray with lid. Protect from light.
      7. Place slides on a vertical plastic staining rack submerged into PBS. Wash slides by moving the slide rack up and down several times. Repeat three times for 5 min each. Protect from light.
      8. Check the immunofluorescence signal under the microscope. Positive immunofluorescence signal consists of a bright green detection of the antigen (Figure 3).
      9. Counterstain nuclei with DAPI solution (see Recipes) for 10 min. Protect from light.
      10. Place slides on a vertical plastic staining rack submerged into PBS. Wash slides by moving the slide rack up and down several times. Repeat three times for 5 min each. Protect from light.
      11. Rinse with Milli-Q water and let dry.
      12. Coverslip brain sections with Fluoro-gel with Tris buffer mounting medium. Let dry.


        Figure 3. Representative confocal images of RNAscope® fluorescent assay using a specific probe to detect TGFβ combined (red) with DAPI staining for nuclei (blue) and immunostaining for microglia/macrophages cells (Iba-1) in the cortex after TBI. Orthogonal projections confocal images and scale bars for A and B (50 μm) and C-E (20 μm).

Data analysis

Details regarding quantification of immunofluorescence and in situ mRNA hybridization has been provided by (Allen et al., 2016; Barrett et al., 2017; Villapol et al., 2017). Briefly:

  1. Positive in situ mRNA hybridization signal consists of a punctate signal representing a single mRNA transcript (Wang et al., 2012).
  2. Take images using a Leica SP8 confocal microscope at 10x and 20x magnification. Take 3-6 microscopic fields within the brain area of interest. All microscope and camera settings (i.e., light level, exposure, gain, etc.) should be identical for all images.
  3. Assign the color label to far red (excitation 647 nm, emission 690 ± 10 nm) for mRNA hybridization signal. Assign the color label to green for the antigen of interest.
  4. Analyze the images with AxionVision software and quantify the hybridization signal with ImageJ64 software (National Institute of Health).
  5. Manually count the number of mRNA-positive cells that colocalize with DAPI nuclei that represent the number of positive cells detected with the antigen of interest. The negative probe used as a control should not contain any stained cells.
  6. Crop and resize images using Adobe Photoshop CS5 if necessary.
  7. Apply changes in brightness and contrast equally to the entire image, and all corresponding images within the same dataset.
  8. Analyze data with a two-way analysis of variance (ANOVA) with Bonferroni post hoc test using GraphPad Prism software v. 5.0 (GraphPad Software, Inc., La Jolla, CA). The two independent variables were sex (female vs. male) and time-point (e.g., 4 h, 1, 3, 7 and 30 days after TBI). A P value < 0.05 was considered statistically significant. Express all figures and tables as mean ± SEM.

Notes

  1. Mounted brain sections can be stored in 100% EtOH following the serial dehydration procedure. At this stage, slides can be stored at -20 °C for a week.
  2. In every pretreatment and amplification step make sure the brain section is covered in solution. We work with mouse brain sections; therefore, bigger tissue might need more volume of solution to cover the section. Not doing so will result in higher noise signal.
  3. Keep the humidity tray warm during the assay.
  4. Because it is important for the tissue to remain wet or humid during the in situ hybridization procedure, it is recommended to work in sets.
  5. At all times check that the integrity of the hydrophobic barrier is not compromised.
  6. All reagents were stored as recommended by the manufacturers.

Recipes

  1. Antifreeze solution
    30% (v/v) glycerol
    30% (v/v) ethylene glycol
    40% (v/v) 0.01 M PBS
  2. 1x wash buffer
    1.47 L ultra-pure water (e.g., Milli-Q water)
    30 ml of 50x wash buffer
    Mix well
  3. 1x antigen retrieval solution (previously known as pretreatment solution 2)
    35 ml of 10x antigen retrieval solution
    315 ml of ultra-pure water
    Mix well and bring to boil (20 min to reach boiling)
  4. DAPI solution
    Dilute DAPI in PBS to a 1:50,000 solution
    Vortex thoroughly. Failing to do so, will result in blue precipitate on the tissue

Acknowledgments

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.

References

  1. Allen, M., Ghosh, S., Ahern, G. P., Villapol, S., Maguire-Zeiss, K. A. and Conant, K. (2016). Protease induced plasticity: matrix metalloproteinase-1 promotes neurostructural changes through activation of protease activated receptor 1. Sci Rep 6: 35497.
  2. Barrett, J. P., Henry, R. J., Villapol, S., Stoica, B. A., Kumar, A., Burns, M. P., Faden, A. I. and Loane, D. J. (2017). NOX2 deficiency alters macrophage phenotype through an IL-10/STAT3 dependent mechanism: implications for traumatic brain injury. J Neuroinflammation 14(1): 65.
  3. Grabinski, T. M., Kneynsberg, A., Manfredsson, F. P. and Kanaan, N. M. (2015). A method for combining RNAscope in situ hybridization with immunohistochemistry in thick free-floating brain sections and primary neuronal cultures. PLoS One 10(3): e0120120.
  4. Villapol, S., Loane, D. J. and Burns, M. P. (2017). Sexual dimorphism in the inflammatory response to traumatic brain injury. Glia 65(9): 1423-1438.
  5. Wang, F., Flanagan, J., Su, N., Wang, L. C., Bui, S., Nielson, A., Wu, X., Vo, H. T., Ma, X. J. and Luo, Y. (2012). RNAscope: a novel in situ RNA analysis platform for formalin-fixed, paraffin-embedded tissues. J Mol Diagn 14(1): 22-29.

简介

小神经胶质细胞和巨噬细胞是响应神经炎症过程的细胞因子的主要生产者。但是这些细胞因子也是由其他神经胶质细胞,内皮细胞和神经元产生的。鉴定产生这些细胞因子的细胞以靶向其不同水平的活化是至关重要的。我们使用双RNAscope荧光原位杂交(FISH)和免疫组织化学(IHC)技术来观察小胶质细胞/巨噬细胞中促炎细胞因子和抗炎细胞因子的mRNA表达模式细胞。使用这些方法,我们可以联合一个mRNA与特定的细胞类型时,通过免疫荧光与不同的细胞标志物结合。来自RNAscope探针的结果IL-1β,TNFα,TGFβ,IL-10或Arg1显示与小胶质细胞/巨噬细胞抗体的共定位。这些靶标探针显示出足够的灵敏度和特异性来检测mRNA表达。新的FISH检测技术结合免疫组化技术将有助于共同确定蛋白质和mRNA的定位,以及提供可靠的mRNA表达水平的量化。
【背景】mRNA原位杂交技术是一种有用的工具,其允许以细胞依赖性方式特异性和选择性标记脑切片中的RNA序列(Grabinski等人,2015 )。此外,由于检测技术的限制,针对这些特定细胞因子的抗体的使用可以产生可变的结果。即,由于这些细胞因子以低丰度表达,因此检测限度成为使用抗体的限制因素。最后,荧光原位杂交(FISH)结合免疫组织化学(ISH)允许以高选择性和特异性检查不同细胞中的细胞因子mRNA谱,从而使我们能够确定细胞因子产生的精确细胞来源TBI之后。该协议描述了如何结合FISH和免疫荧光成像可以跨越mRNA和蛋白质分析之间的差距。我们可以鉴定小胶质细胞/巨噬细胞产生的目标mRNA。同一组织中RNA和蛋白质表达的分析允许区分小胶质细胞/巨噬细胞和其他细胞类型的细胞特异性产生。关于性别对创伤性脑损伤(TBI)后的炎症特征的影响的研究是有限的。在我们最近的出版物(Villapol等人,2017)中,我们使用了RNAscope技术与免疫荧光技术相结合来确定促炎症(例如, IL1β和TNFα)以及雄性和雌性小鼠受伤脑中小胶质细胞/巨噬细胞中的抗炎症(例如,TGFβ和Arg1)细胞因子mRNA表达谱。我们的数据表明,在TBI后的第一周,小胶质细胞/巨噬细胞中发生促炎和抗炎细胞因子表达的混合模式。此外,我们先前已经显示NOX2 - / - 小鼠受损皮层中的小胶质细胞/巨噬细胞中的IL-10水平显着增加(Barrett等人,2017) 。

总之,当检测不同细胞中的细胞因子mRNA时,FISH的使用提高了特异性和敏感性,这通过对小神经胶质细胞/巨噬细胞的抗体免疫染色来证实。这种方法使我们能够鉴定脑损伤后细胞因子产生的细胞来源,其置信度大大提高。

关键字:原位杂交, RNAscope, 创伤性脑损伤(TBI), 巨噬细胞, 免疫荧光, 小胶质细胞, 神经炎症, 细胞因子

材料和试剂

  1. 厚的沃特曼纸(Fischerbrand 色谱纸)(Fisher Scientific,目录号:05-714-4)
  2. 明胶包被的载玻片(Superfrost Plus)(Fisher Scientific,目录号:12-550-15)
  3. 显微镜盖玻璃(24×50mm)(Thermo Fisher Scientific,Thermo Scientific TM,产品目录号:3422ERI)
  4. ImmEdge疏水屏障笔(Advanced Cell Diagnostics,目录号:310018)
  5. Kimwipes(KCWW,Kimberly-Clark,目录号:34133)
  6. 铝箔纸
  7. 小鼠(来自THE JACKSON LABORATORY的C57BL / 6,目录号:000664)
  8. RNAscope阳性对照探针-Mm-Ppib(肽基脯氨酰异构酶B)(Advanced Cell Diagnostics,目录号:313911)
  9. RNAscope阴性对照探针-DapB(Advanced Cell Diagnostics,目录号:310043)
  10. RNAscope探针-Mm-Tgfβ1(转化生长因子β1)(Advanced Cell Diagnostics,目录号:407751)
  11. RNAscope探针-Mm-IL1β(白细胞介素1β)(Advanced Cell Diagnostics,目录号:316891)
  12. RNAscope探针-Mm-TNFα(肿瘤坏死因子α)(Advanced Cell Diagnostics,目录号:311081)
  13. RNAscope探针-Mm-Arg1(精氨酸酶1)(Advanced Cell Diagnostics,目录号:403431)
  14. 磷酸盐缓冲盐水(PBS),10倍溶液(Fisher Scientific,目录号:BP39920)
  15. 多聚甲醛(PFA)储备液制备4%(w / v)PFA,PRILLS(电子显微镜科学,目录号:19200)
  16. 蔗糖结晶储备液(Fisher Scientific,目录号:S5)制备在超纯水中稀释的30%(w / v)溶液
  17. 超纯水(如,Milli-Q)
  18. 在超纯水(例如,Milli-Q水)中新鲜制备的50%(v / v)乙醇溶液
  19. 在超纯水(例如Milli-Q水)溶液中新鲜制备的70%(v / v)乙醇
  20. 无水乙醇(200级乙醇)(Fisher Scientific,目录号:BP28184)
  21. RNAscope H 2 O 2 2&lt;蛋白酶加试剂(Advanced Cell Diagnostics,目录号:322330)
  22. RNAscope 2.5 HD检测试剂-REP(Advanced Cell Diagnostics,产品目录号:322360)(阅读注释1)
  23. RNAscope®目标检索试剂(Advanced Cell Diagnostics,目录号:322000) - 以前称为预处理2溶液
  24. 正常山羊血清(NGS)封闭液(Vector Laboratories,目录号:S-1000)
  25. Triton X-100
  26. 多克隆抗兔Iba-1(离子钙结合适配体分子-1)抗体(Wako Pure Chemical Industries,目录号:019-19741)
  27. 多克隆抗兔P2Y12抗体(AnaSpec,目录号:AS-55042A)
  28. 多克隆抗大鼠F4 / 80抗体(R&amp; D systems,目录号:MAB5580)
  29. 山羊抗兔Alexa Fluor 488二抗(Thermo Fisher Scientific,目录号:A-11034)
  30. 山羊抗大鼠Alexa Fluor 488二抗(Thermo Fisher Scientific,目录号:A-11006)
  31. 氟胶(含Tris缓冲液)固定介质,20ml(电子显微镜科学,目录号:17985-10)
  32. 甘油
  33. 乙二醇
  34. RNAscope洗涤缓冲液试剂(4×60ml)(Advanced Cell Diagnostics,目录号:310091)
  35. 4',6-二脒基-2-苯基吲哚(DAPI)溶液(Sigma-Aldrich,目录号:D9542)
  36. 防冻液(见食谱)
  37. 1倍洗涤缓冲液(见食谱)
  38. 1x抗原修复液(见食谱)
  39. DAPI解决方案(请参阅食谱)

设备

  1. Gilson TM PIPETMAN Classic TM吸管(Gilson,型号:P20,P200,P1000,目录号:F123600,F123601,F123602)
  2. 2升烧杯
  3. 超长镊子(Thermo Fisher Scientific,Fisherbrand TM,目录号:10-316A)
  4. 定时器,可溯源TM Nano TM(Fisher Scientific,Fisherbrand TM,产品目录号:14-649-83)
  5. 幻灯片持有人手柄,24幻灯片(电子显微镜科学,目录号:62543-06)
  6. 滑动切片机MicromHM 430(Thermo Fisher Scientific,Thermo Scientific TM,型号:HM 430,目录号:910010)
  7. HybEz TM杂交系统(Advanced Cell Diagnostics,目录号:310010)
  8. ACD HybEZ TM带有盖子的湿度控制盘(Advanced Cell Diagnostics,产品目录号:310012)
  9. 用EasyDipTM载玻片染色(Electron Microscopy Sciences,目录号:62540-01)组织Tek载玻片染色组
  10. ACD EZ-slide holder / rack(Advanced Cell Diagnostics,产品目录号:310017)
  11. 带有磁力搅拌器的Thermix 210T型电热板(Fisher Scientific,型号:210T型,目录号:11-493-210T)
  12. 使用Photometrics相机(CoolSNAP,fx,Axioskop,Roper Scientific,序列号:A02M86017)在Axioplan 2显微镜(Carl Zeiss,型号:Axioplan 2,目录号:451485)上获取荧光图像。
  13. 徕卡SP8共聚焦显微镜(徕卡显微系统,型号:徕卡TCS SP8)

软件

  1. GraphPad Prism软件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。,授权给:3018897(Carl Zeiss MicroImaging,GmbH)
  4. ImageJ64软件(国家卫生研究院)

程序

使用RNAscope技术2.5红色荧光试剂盒对新鲜冷冻组织进行荧光原位杂交,并进行了一些修改。
所有目标探针由20个短的双Z寡核苷酸探针对组成,这些探针对是基因特异性的,并从ACD获得。

  1. RNAscope探针-Mm-Arg1(登录号NM_007482.3,目标区2-1114)
  2. RNAscope探针-Mm-TNFα(登录号NM_013693.2,目标区域41-1587)
  3. RNAscope探针-Mm-Tgfβ1(登录号NM_011577.1,目标区域588-1913)
  4. RNAscope探针-Mm-IL1β(登录号NM_008361.3,目标区域2-950)
  5. RNAscope阳性对照探针-Mm-Ppib(登录号NM_011149.2,目标区域98-856)
  6. RNAscope®阴性对照探针-DapB(针对细菌基因)

  1. 组织
    1. 如前所述(Villapol等人,2017),用CO 2安乐死小鼠并用冰冷的磷酸盐缓冲盐水(PBS)经心脏灌注。

    2. 用4%(v / v)PFA整夜固定整个大脑
    3. 将小鼠脑转移到PBS中的30%(v / v)蔗糖溶液中,在4℃下脱水48小时,或者直到大脑完全下沉到管底部。

    4. 使用滑动切片机将20μm厚的小鼠脑冠状切片切片
    5. 在防冻液中冷冻保护大脑(见食谱),并保存在-20°C。

  2. 在开始之前
    1. 准备1.5升的1倍洗涤缓冲液(见食谱)。
    2. 将HybEZ TM 杂交系统设置为40°C。
    3. 剪下Whatman纸,放在HybEZ TM湿纸盘底部的中央,加入约50毫升的超纯水。
    4. 温热HybEZ TM TM湿度控制托盘,使用前应将湿的Whatman纸放置30分钟。

    5. 在使用前允许“Pretreat 1”溶液(过氧化氢溶液)在室温(RT)下平衡

    6. 在RT下平衡扩增试剂(AMP溶液1-6,从ACD获得)
    7. 温度控制和目标探头在40°C下10分钟,在使用前在室温下冷却。

  3. 用于荧光原位杂交(FISH)的RNAscope 技术
    1. 装载明胶涂层幻灯片上的冠状脑部分(20微米厚),并储存在-80°C,直到使用。尝试找到幻灯片底部的大脑部分,以避免在清洗过程中使用过量的缓冲液。
    2. 将玻片放在垂直的塑料染色架上,浸入50%新鲜乙醇溶液中5分钟。
    3. 将包含载玻片的垂直塑料染色架转移到含有70%乙醇溶液的EasyDip TM载玻片染色系统中5分钟(图1A和视频1)。
    4. 将含有载玻片的垂直塑料染色架转移到含有100%乙醇溶液的EasyDip TM载玻片染色系统中5分钟。
    5. 将含有载玻片的垂直塑料染色架转移到含有第二个100%乙醇溶液的EasyDip TM载玻片染色系统中5分钟(请参阅注释1)。
    6. 从100%乙醇中取出载玻片,在载玻片架上放置30分钟。

    7. 使用immEdge疏水屏障笔(图1B和视频1)在每个部分周围绘制疏水屏障
    8. 从这一步开始,不要让大脑部分完全变干。
    9. 预处理1(过氧化氢处理)
      1. 将载玻片放在ACD EZ载玻片架上,并在每个切片上添加60-100μl的预处理溶液1(图1C和视频1,请参阅注释2)。
      2. 在RT预处理溶液1中孵育脑部分10分钟。将预处理解决方案1添加到第一张幻灯片后启动计时器。
      3. 摒弃预处理解决方案1,将幻灯片滑入废物容器中,或者在必要时敲击Kimwipe上的幻灯片侧面。
      4. 将幻灯片放入浸在超纯水中的垂直塑料染色架中。

      5. 上下移动染色架以清洗载玻片
    10. 预处理2(抗原修复液)
      1. 将350毫升1x抗原修复液(预处理溶液2)(见食谱)用热板煮沸。 (通常需要40分钟才能煮沸。)建议用铝箔纸覆盖容器。
      2. 检查确保温度在100-104°C之间。

      3. 使用长弧形钳子,将载玻片的垂直塑料染色架慢慢浸入煮沸的抗原修复液中。
      4. 检查确保温度在100-104°C之间。
      5. 煮的幻灯片2至10分钟(这取决于组织的质量)。
      6. 用超纯水冲洗载玻片(重复一次)。
    11. 预处理3(蛋白酶消化)
      1. 将幻灯片滑入废物容器中,或者在必要时敲击Kimwipe上的幻灯片侧面,以滗去超纯水。
      2. 将幻灯片放在ACD EZ滑轨架上。
      3. 每个脑组添加约60-100μl预处理溶液3(蛋白酶溶液)以覆盖整个组织切片(图1D和视频1,请参阅注释2)。
      4. 将ACD EZ载玻片固定器/支架放入预热的HybEZ TM带盖盖的烤箱中,在40°C孵育30分钟。
      5. 拆下ACD EZ滑块支架/支架,然后将ACD HybEZ TM 湿度控制托盘放回HybEZ TM烘箱杂交系统(图1E和视频1)。< br />

        图1. RNAscope方案的组成部分。 :一种。在蒸馏水中洗涤; B.在每个部分周围绘制疏水屏障; C.加入预处理溶液; D.扩增溶液; E.持有人/机架。

        视频1

      6. 如果需要的话,将幻灯片滑入废物容器中或者通过敲击Kimwipe上的幻灯片的侧面来滗析预处理溶液3。
      7. 将幻灯片放入浸在超纯水中的垂直塑料染色架中。

      8. 上下滑动滑动架来清洗滑梯
    12. 目标探针杂交
      1. 将幻灯片滑入废物容器中,或者在必要时敲击Kimwipe上的幻灯片侧面,以滗去超纯水。
      2. 将幻灯片放在ACD EZ滑片架上(请参阅注3)。

      3. 每个脑区添加60-100μl的目标探针或对照探针以覆盖整个组织切片(请参阅注释2)
      4. 将ACD EZ载玻片固定器/支架放入预热的ACD HybEZ TM 湿度控制盘中,并在HybEZ TM 杂交系统中于40°C孵育2小时。
      5. 卸下ACD EZ滑块支架/支架,然后将ACD HybEZ TM 湿度控制托盘放回到HybEZ TM 杂交系统中。
      6. 通过滑动幻灯片到废物容器中或通过敲击Kimwipe上的幻灯片边来滗析控制探头和目标探头。
      7. 将载玻片放入浸在1倍清洗缓冲液中的垂直塑料染色架中。
      8. 将载玻片架上下移动数次,清洗载玻片2分钟。
    13. 放大步骤(AMP 1-AMP 6)
      1. 通过将幻灯片滑动到废物容器中或通过敲击Kimwipe上的幻灯片的侧面来滗析1x清洗缓冲液。
      2. 将幻灯片放在ACD EZ载玻片架上,不要让组织片完全干燥。

      3. 在每个组织切片中加入60-100μl每个脑切片的AMP溶液(请参阅注2)
      4. 将ACD EZ载玻片固定器/支架放入ACD HybEZ TM 湿度控制盘中,根据表1进行培养。

        表1.扩增溶液及其相应的孵育时间和温度


      5. 通过将幻灯片滑入废物容器或轻敲Kimwipe上幻灯片的侧面来滗析AMP解决方案。
      6. 将玻片放在垂直的塑料染色架上,浸入1x洗涤缓冲液中。
      7. 将载玻片架上下移动数次,清洗载玻片2分钟。
    14. 检测信号(室温10分钟)
      1. 每张载玻片上混合60μl红色-A和1μl红色-B溶液。避光。
      2. 通过将幻灯片滑动到废物容器中或通过敲击Kimwipe上的幻灯片的侧面来滗析1x清洗缓冲液。
      3. 将幻灯片放在ACD EZ载玻片架上,不要让组织片完全干燥。
      4. 每个组织部分添加60μL的检测解决方案(阅读注2)。确保从正面和负面的控制幻灯片开始。
      5. 在带有盖子的ACD HybEZ TM 湿度控制盘内,在室温下孵育10分钟。避光。将检测解决方案添加到第一张幻灯片后立即启动计时器。
      6. 通过比较阳性对照和阴性对照,每隔两分钟检查杂交信号的进展。阳性对照应该是红色的,而阴性对照应该显示很少或没有背景。
      7. 通过将幻灯片滑入废物容器或敲击Kimwipe上幻灯片的侧面来滗析检测解决方案。
      8. 将幻灯片放置在浸入超纯水中的垂直塑料染色架上。
      9. 将载玻片架上下移动数次,清洗载玻片2分钟。
      10. 在进行免疫组织化学之前,在显微镜下检查杂交信号。阳性杂交信号由表示mRNA转录物的几个点状信号(红色点)组成(Wang等人,2012)(图2)。


        图2.在选择的脑切片上的阳性对照探针和细胞核(DAPI,蓝色)的代表性图像比例尺= 50μm。

    15. 免疫荧光
      1. 将含有载玻片的垂直塑料染色架转移到含有PBS的EasyDip TM载玻片染色系统中。
      2. 通过上下移动滑动架多次清洗滑道。重复三次。
      3. 在补充有0.3%(v / v)Triton X-100(PBST)的PBS中的5%(v / v)正常山羊血清(NGS)中在室温下封闭1小时(或在4℃过夜)在ACD HybEZ带有盖子的TM湿度控制盘。
      4. 在PBST中的5%(v / v)NGS中的一级抗体中孵育过夜,在以下稀释度下在4℃下过夜:
        多克隆抗兔Iba-1(1:500)
        多克隆抗兔P2Y12(1:1,000)
        多克隆抗大鼠F4 / 80(1:200)
      5. 将幻灯片放在淹没在PBS中的垂直塑料染色架上。通过上下移动滑动架多次清洗滑道。重复三次,每次5分钟。
      6. 在ACD HybEZ TM 中室温孵育相应的Alexa Fluor 488二抗(PBST中5%(v / v)NGS中1:1,000) >带有盖子的湿度控制盘。避光。
      7. 将幻灯片放在淹没在PBS中的垂直塑料染色架上。通过上下移动滑动架多次清洗滑道。重复三次,每次5分钟。避光。
      8. 在显微镜下检查免疫荧光信号。阳性免疫荧光信号由鲜绿色的抗原检测组成(图3)。
      9. 用DAPI溶液反击细胞核(见食谱)10分钟。避光。
      10. 将幻灯片放在淹没在PBS中的垂直塑料染色架上。通过上下移动滑动架多次清洗滑道。重复三次,每次5分钟。避光。
      11. 用Milli-Q水冲洗干净。
      12. 用含有Tris缓冲液的Fluoro-gel封闭脑切片。让干。


        图3.使用特异性探针检测TGFβ结合(红色)和DAPI染色核(蓝色)和免疫染色的RNAscope荧光测定的代表性共焦图像TBI后皮层内的小胶质细胞/巨噬细胞(Iba-1) A和B(50μm)和C-E(20μm)的正交投影共聚焦图像和比例尺。

数据分析

(Allen等人,2016; Barrett等人,2017; Barrett等人,2017)提供了关于定量免疫荧光和原位mRNA杂交的细节。 Villapol 等,2017)。简述:

  1. 阳性原位mRNA杂交信号由代表单个mRNA转录物的点状信号组成(Wang et al。,2012)。
  2. 用10倍和20倍放大倍率的Leica SP8共聚焦显微镜拍摄图像。在感兴趣的大脑区域内取3-6个显微镜视野。所有的显微镜和照相机设置(即,
  3. 将颜色标签分配到远红色(激发647nm,发射690±10nm)用于mRNA杂交信号。将感兴趣抗原的颜色标签指定为绿色。
  4. 用AxionVision软件分析图像,并用ImageJ64软件(National Institute of Health)量化杂交信号。
  5. 手动计数与DAPI细胞核共同定位的mRNA阳性细胞的数量,代表用感兴趣的抗原检测的阳性细胞的数量。用作对照的阴性探针不应含有任何染色的细胞。

  6. 使用Adobe Photoshop CS5裁剪和调整图像大小
  7. 将亮度和对比度的变化同样应用于整个图像以及同一数据集内的所有对应图像。
  8. 使用GraphPad Prism软件v.5.0(GraphPad Software,Inc.,La Jolla,CA),用Bonferroni事后检验用双因素方差分析(ANOVA)分析数据。两个独立变量是性别(女性对男性)和时间点(例如,在TBI后4小时,1天,3天,7天和30天)。 A&lt; em&gt; P&lt; em&gt;值&lt; 0.05被认为有统计学意义。用平均值±SEM表示所有的数字和表格。

笔记

  1. 安装的脑切片可以在连续脱水程序后储存在100%EtOH中。在这个阶段,幻灯片可以储存在-20°C一个星期。
  2. 在每个预处理和放大步骤中,确保大脑部分覆盖在溶液中。我们使用鼠标大脑部分,因此,较大的组织可能需要更多体积的溶液来覆盖该部分。不这样做会导致更高的噪音信号。
  3. 分析过程中保持湿度盘温热。
  4. 因为在原位杂交过程中组织保持湿润或潮湿是重要的,所以建议成组工作。
  5. 始终检查疏水屏障的完整性是否受损。
  6. 所有试剂均按制造商推荐的方式储存。

食谱

  1. 防冻液
    30%(v / v)甘油
    30%(v / v)乙二醇
    40%(v / v)0.01M PBS
  2. 1次洗涤缓冲液
    1.47L超纯水(例如,Milli-Q水)
    30毫升的50倍洗涤缓冲液
    混合好
  3. 1x抗原修复液(以前称为预处理液2)
    35毫升的10倍抗原修复液

    315毫升的超纯水 充分混合,煮沸(20分钟煮沸)
  4. DAPI解决方案
    将PBS中的DAPI稀释到1:50,000的溶液。
    彻底涡旋。如果不这样做,将导致组织蓝色沉淀

致谢

这项工作得到了美国国立卫生研究院拨款R03NS095038(S. Villapol),R01NS067417(M.P. Burns)和乔治敦大学医学中心(I. Mocchetti)提供的生物医学研究院院长(Toulmin试点项目)的支持。我们要感谢Kathy Maguire-Zeiss博士让我们使用她的荧光显微镜,并感谢Lucas Djavaherian在录像中的帮助。该协议已经被改编自(Barrett等人,2017,Villapol等人,2017)。作者声明,研究是在没有任何商业或财务关系的情况下进行的,可能被认为是潜在的利益冲突。

参考

  1. Allen,M.,Ghosh,S.,Ahern,G. P.,Villapol,S.,Maguire-Zeiss,K.A。和Conant,K.(2016)。蛋白酶诱导的可塑性:基质金属蛋白酶-1通过激活蛋白酶活化的受体1促进神经结构改变。 a> Sci Rep 6:35497。
  2. Barrett,J.P.,Henry,R.J.,Villapol,S.,Stoica,B.A.,Kumar,A.,Burns,M.P.,Faden,A.I。和Loane,D.J。(2017)。 NOX2缺乏症通过IL-10 / STAT3依赖性机制改变巨噬细胞表型:影响创伤性脑损伤。 J Neuroinflammation 14(1):65.
  3. Grabinski,T.M.,Kneynsberg,A.,Manfredsson,F.P。和Kanaan,N.M。(2015)。 将RNAscope原位杂交与免疫组织化学结合的方法,漂浮的大脑切片和主要的神经元文化。 PLoS One 10(3):e0120120。
  4. Villapol,S.,Loane,D.J。和Burns,M.P.(2017)。 对创伤性脑损伤的炎症反应中的性别二态性 65(9):1423-1438。
  5. Wang,F.,Flanagan,J.,Su,N.,Wang,L.C.,Bui,S.,Nielson,A.,Wu,X.,Vo,H.T.,Ma,X.J。和Luo,Y。(2012) RNAscope:一种新颖的原位RNA分析平台,用于福尔马林固定,石蜡包埋的组织。 J Mol Diagn 14(1):22-29。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Lanfranco, M., 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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