Method Article

Detection of Axonally Localized mRNAs in Brain Sections Using High-Resolution In Situ Hybridization

DOI:

10.3791/52799

⸱

June 17th, 2015

 ,  ,  , 
1College of Physicians and Surgeons, Columbia University

In This Article

Summary

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

RNA in situ hybridization (ISH) enables the visualization of RNAs in cells and tissues. Here we show how combination of RNAscope ISH with immunohistochemistry or histological dyes can be successfully used to detect mRNAs localized to axons in sections of mouse and human brains.

Abstract

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

mRNAs are frequently localized to vertebrate axons and their local translation is required for axon pathfinding or branching during development and for maintenance, repair or neurodegeneration in postdevelopmental periods. High throughput analyses have recently revealed that axons have a more dynamic and complex transcriptome than previously expected. These analysis, however have been mostly done in cultured neurons where axons can be isolated from the somato-dendritic compartments. It is virtually impossible to achieve such isolation in whole tissues in vivo. Thus, in order to verify the recruitment of mRNAs and their functional relevance in a whole animal, transcriptome analyses should ideally be combined with techniques that allow the visualization of mRNAs in situ. Recently, novel ISH technologies that detect RNAs at a single-molecule level have been developed. This is especially important when analyzing the subcellular localization of mRNA, since localized RNAs are typically found at low levels. Here we describe two protocols for the detection of axonally-localized mRNAs using a novel ultrasensitive RNA ISH technology. We have combined RNAscope ISH with axonal counterstain using fluorescence immunohistochemistry or histological dyes to verify the recruitment of Atf4 mRNA to axons in vivo in the mature mouse and human brains.

Introduction

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

Axonal mRNA recruitment and local translation enable axons to respond to extracellular stimuli in a temporally and spatially acute manner1. Intra-axonal protein synthesis is best understood in the context of neurodevelopment where it plays crucial roles in growth cone behavior2-8, axon pathfinding9-11 and retrograde signaling12,13. But far less is known about the functional significance of axonal protein synthesis in post-developmental neurons when axonal mRNA and ribosome levels are greatly reduced14,15. Mature vertebrate axons have long been thought to be translationally inactive16. However, recent studies indicate that local translation is reactivated in mature axons under pathological conditions. For instance, a subset of mRNAs is recruited to regenerating axons following nerve injury and intra-axonal protein synthesis is required for the correct regeneration of these axons17. Additionally, our group has demonstrated that specific mRNAs are recruited to axons after local exposure to the Alzheimer’s disease peptide Aβ1-42, and local translation of the transcription factor ATF4 is required to propagate the neurodegenerative effects of Aβ1-42 from axons to the neuronal soma18. Finally, high throughput analyses have revealed that mature axons have a more complex and dynamic transcriptome than expected18-21, especially under pathological conditions. In light of these studies, a highly sensitive and specific method to detect axonally localized mRNAs in the adult nervous system is needed.

Much of the work on mRNA recruitment and local translation in mature axons has been performed on cultured neurons. This is especially true for transcriptome analyses since specialized culturing methods exist that allow the isolation of axons from the somato-dendritic compartment18-20. Although such studies have given valuable insight into the role of local translation in mature axons, the question whether cultured neurons faithfully represent the situation in vivo or if mRNA recruitment is an adaptive response of axons to culturing conditions is still open. Few studies have provided evidence of mRNA recruitment to mature axons in vivo. For example, the transcript coding for the olfactory marker protein has been detected in axons in adult sensory neurons22. A transgene containing the 3’ UTR of β-actin mRNA is transported to axons in peripheral and central nervous system neurons in mice and is locally translated after developmental periods23. Lamin b2 mRNA is localized to retinal axons in Xenopues laevis tadpoles and its depletion affects axon maintenance after axonal development21. Interference with the axonal transport of the mRNA encoding for cytochrome C oxidase IV alters mouse behavior24. Finally, Atf4 mRNA is found in adult axons of mice and human brains in the context of Aβ1-42-induced neurodegeneration18.

High throughput transcriptome analyses have proven to be useful to identify mRNA profiles in isolated axons in vitro but have limitations for in vivo studies since in whole tissues axons are never found in isolation but intermingled with neuronal cell bodies, glial cells and other cell types. Thus, such analyses have to be combined with imaging techniques that confirm the subcellular localization of mRNAs. RNA in situ hybridization (RNA ISH) allows the detection and visualization of specific RNA sequences in cells and tissues. However, original RNA ISH assays were suitable only for the identification of highly abundant RNAs25, which is rarely the case for axonally localized mRNAs. For the last decades increasing efforts have been put into developing novel technologies that allow the detection of mRNAs at the single-molecule level25,26. For instance, Singer and colleagues have developed ISH probes to detect mRNAs in single cells, consisting in 5 non-overlapping fluorescently labelled 50-mers (for details refer to 27). The main difference between the above mentioned techniques and the one here described is that the later uses 20 double Z-structure (not linear) probes that typically target ~ 1 kb region of the RNA of interest ensuring specificity and low background levels. Probes are then hybridized with preamplifier and amplifier sequences that are finally fluorescently labeled or conjugated with enzymes that allow chromogenic reactions. These amplification steps improve the signal-to-noise ratio compared to other ISH technologies28. Here we describe two protocols using RNAscope combined either with fluorescence immunocytochemistry or with histological dyes allowing axonal counterstaining. Both protocols are suitable to visualize axonal localization of Atf4 mRNA in adult mouse and human brains.

Access restricted. Please log in or start a trial to view this content.

Protocol

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

All animal procedures were approved by the IACUC of Columbia University and applicable guidelines for the care and use of laboratory animals were followed. Note: Prepare all buffers used for the ISH procedures in RNase-free or DEPC-treated water. This recommendation is not strictly necessary after ISH has been completed but it is suggested that buffers are still prepared in autoclaved double-distilled water and/or sterilized by filtering.

1. Detection of Atf4 mRNA Localized to Cholinergic Axons in the Adult Mouse Brain using Fluorescence In Situ Hybridization (FISH) Followed by Immunohistochemistry

  1. Sample preparation for paraformaldehyde-fixed frozen brain slices
    1. For the following example, prepare slices from fixed frozen mouse brains.
      1. Briefly, sacrifice 9 month-old mice by anesthesia with ketamine (100 mg kg-1 body weight) and xylazine (10 mg kg-1 body weight) followed by transcardial infusion of 4% paraformaldehyde in PBS (pH 7.4).
      2. Remove brains and post-fix in 4% paraformaldehyde for 24 hrs at 4 oC.
      3. After fixation, wash samples three times in PBS and cryoprotect in 30% sucrose in PBS (pH 7.4) for 24 hrs at 4 oC.
      4. Embed brains in OCT freezing compound, serially section at 20 µm on a cryostat and mount on poly-D-lysine treated microscope slides. Keep brain slices at -20 oC until use.
    2. Remove paraformaldehyde-fixed brain slices from freezer and air-dry for 30 min at RT.
    3. Wash three times with 1x PBS.
    4. In a fume hood, re-fix brain slices 20 min with 4% paraformaldehyde at RT.
      ​NOTE: CAUTION: 4% paraformaldehyde is toxic and must be handled and discarded appropriately following safety protocols.
    5. Wash three times with 1x PBS.
    6. Dehydrate brain slices.
      1. Prepare 50%, 75% and 100% ethanol solutions.
      2. Immerse slides in 50% ethanol for 5 min at RT.
      3. Immerse slides in 75% ethanol for 5 min at RT.
      4. Immerse slides in 100% ethanol for 5 min at RT.
      5. Immerse slides in fresh 100% ethanol for 5 min at RT. Note: Alternatively, slides can be stored in 100% ethanol at -20oC up to 1 month.
  2. FISH assay followed by immunohistochemistry using heat-induced antigen/RNA unmasking
    1. Before starting prepare all required material:
      1. Heat the hybridization oven to 40 oC and place a tray containing distilled water in the oven to create a humidified environment.
      2. Take a slide box and place paper towels soaked in distilled water inside to humidify the slide box. Place the slide box in the hybridization oven.
      3. Remove probes (both negative and target probes) from fridge and heat them in the hybridization oven for 10 min. Let the probes cool down at RT.
      4. Equilibrate amplification reagents AMP 1-4 (included in the Fluorescent Multiplex Reagent Kit) at RT.
      5. Prepare antigen retrieval solution: 10 mM sodium citrate (pH 6), 0.05% Tween 20. Note: Solution can be stored at RT indefinitely and be used for future staining.
      6. Prepare 1x wash buffer diluting the 50x stock solution (included in the Fluorescent Multiplex Reagent Kit) in RNase-free or DEPC-treated water.
        NOTE: 1x wash buffer can be stored at RT for 1 month and be used for future staining. 50x wash buffer might precipitate. If so, heat 50x wash buffer at 40 oC for 10 min before preparing the 1x solution.
    2. Remove slides from 100% ethanol and air-dry for 5 min at RT.
    3. Prepare a coplin jar containing 50 ml of antigen retrieval solution. Immerse slides in antigen retrieval solution and boil them for 10 min in a microwave oven.
      NOTE: Alternatively place slides horizontally in a 100 mm diameter round glass dish containing 100 ml of retrieval solution.
      1. Fill up a 1 L beaker with distilled water and place it in the microwave.
        NOTE: The water will “buffer” the heat when performing unmasking.
      2. Place the slides in antigen retrieval solution inside the microwave. Boil slides at high power for 5 min. Immediately after samples have stopped boiling, heat slides again at high power for extra 5 min.
      3. Cool down slides at RT.
        NOTE: CRITICAL STEP: boiling duration and procedure should be determined by user, depending on the microwave characteristics. The faster the boiling starts the higher the chances of solution evaporation. In this case it is recommended that samples are boiled for shorter time periods more than twice to complete a total unmasking time of 10 min. On the contrary, if heating is performed at medium or low power, unmasking can be performed once for 10 min. However, if samples do not boil continuously for a total of approximately 10 min, this might result in partial unmasking of both the target RNA and protein to be detected.
    4. Wash slides three times with 1x PBS.
    5. Add 2 - 3 drops (~100 μl) of the dapB probe set to those brain slices used to assess background staining. Note: The dapB probe set targets a bacterial gene and should not recognize any mammalian RNA.
    6. Add 2 - 3 drops of the targeting probe set to those brain slices used to detect the RNA of interest. Note: a probe set targeting residues 20 - 1,381 of the mouse Atf4 mRNA is used in this example.
    7. Cover slides with parafilm to avoid probe evaporation, place them in the humidified slide box inside the hybridization oven and incubate them at 40 oC for 2 hrs.
      ​NOTE: OPTIONAL: additional brain slices can be hybridized with a probe set targeting mouse Polr2A and used as positive controls.
    8. Wash slides twice with 1x wash buffer for 2 min at RT.
    9. Add 2 - 3 drops of amplification reagent AMP 1-FL to each brain slice, cover slides with parafilm, place them in the slide box and incubate at 40 oC for 30 min in the hybridization oven.
    10. Wash slides twice with 1x wash buffer for 2 min at RT.
    11. Add 2 - 3 drops of amplification reagent AMP 2-FL to each brain slice, cover slides with parafilm, place them in the slide box and incubate at 40 oC for 15 min in the hybridization oven.
    12. Wash slides twice with 1x wash buffer for 2 min at RT.
    13. Add 2 - 3 drops of amplification reagent AMP 3-FL to each brain slice, cover slides with parafilm, place them in the slide box and incubate at 40 oC for 30 min in the hybridization oven.
    14. Wash slides twice with 1x wash buffer for 2 min at RT.
    15. Add 2 - 3 drops of amplification reagent AMP 4-FL to each brain slice, cover slides with parafilm, place them in the slide box and incubate at 40 oC for 15 min in the hybridization oven.
    16. Wash slides twice with 1x wash buffer for 2 min at RT and twice with 1x PBS.
    17. Add 100 - 200 μl (or enough volume to completely cover slices) of a blocking solution containing 3 mg/ml BSA, 100 mM glycine and 0.25% Triton X-100 in PBS (pH 7.4) to the slides, cover them with parafilm and incubate for 30 min at RT.
    18. Add 100 - 200 μl of anti-ChAT antibody diluted in blocking solution (1/100) to the slides, cover them with parafilm and incubate for 2 days at 4 oC. Make sure that brain slices don’t dry out. If needed, re-apply anti-ChAT antibody solution to brain slices 24 hrs after incubation.
    19. Wash slides three times in 1x PBS for 5 min at RT.
    20. Add 100 - 200 μl of the appropriate Alexa-conjugated secondary antibody (e.g., Alexa-594 donkey anti-goat for this particular example) to the slides, cover with parafilm and incubate for 1 hr at RT.
    21. Wash slides three times in 1x PBS for 5 min at RT.
    22. Wash slides once with distilled water.
    23. Mount slides with DAPI-containing mounting medium.
    24. Visualize brain slices under a fluorescence microscope.

2. Detection of Atf4 mRNA Localized to Axons in Human Brain Samples using Chromogenic In Situ Hybridization (CISH) Followed by Luxol Fast Blue and Cresyl Violet Counterstaining

  1. Sample preparation for formalin-fixed paraffin-embedded human brain samples
    1. Bake slices in a dry oven at 60 oC for 1 hr.
    2. Deparaffinize brain slices in a fume hood.
       NOTE: CAUTION: reagents are toxic and must be handled and discarded following the appropriate security guidelines
      1. Immerse slides in xylene alternative clearing agent twice for 10 min.
      2. Immerse slides in 100% ethanol twice for 1 min.
      3. Air-dry slices 5 min at RT. Note: Alternatively samples might be dried at RT O/N but must be used within 24 hrs.
  2. Diaminobenzidine (DAB)-based chromogenic ISH assay followed by luxol fast blue and cresyl violet stain using heat-induced and protease-induced RNA unmasking
    1. Before starting prepare required materials:
      1. Heat the hybridization oven to 40 oC and place a tray containing distilled water to create a humidified environment.
      2. Take a slide box and place paper towels soaked in distilled water inside to humidify the slide box. Place the slide box in the hybridization oven.
      3. Prepare 1x Pretreat 2 by diluting the 10x stock solution (included in the CISH Reagent Kit) in RNase-free or DEPC-treated water. If pretreatment is performed on a hot plate, add 100 ml of pretreat solution to a 100 mm diameter glass dish to increase the contact surface between the dish and the hot plate (if pretreatment is performed in a microwave, a coplin jar can be used instead as specified in 1.2.3). Heat solution to 100 oC and keep it boiling no longer than 30 min before submerging samples.
      4. Heat negative and positive probes 10 min at 40 oC and cool down at RT.
      5. Equilibrate amplification reagents AMP 1-6 (included in the CISH Reagent Kit) at RT.
      6. Prepare 1x wash buffer diluting the 50x stock solution (included in the CISH Reagent Kit) in distilled water. Note: 1x wash buffer can be stored at RT for 1 month and be used for future staining.
    2. Add ~4 drops (~120 μl) of Pretreat 1 to each brain slice, cover with parafilm and incubate at RT for 10 min.
    3. Wash 3 to 5 times in fresh distilled water.
    4. Perform heat-induced unmasking:
      1. Transfer slides to hot Pretreat 2 (included in the CISH Reagent Kit) solution and boil for 15 min. Note: Boiling can be performed on a hot plate ensuring continuous boiling or in a microwave following critical steps specified in 1.2.3). 
    5. Immediately transfer slides to distilled water and wash 3 to 5 times.
    6. Wash slides 3 to 5 times in fresh 100% ethanol.
    7. Air-dry slices 5 min at RT. Note: Alternatively slices can be dried O/N.
    8. Perform protease-induced unmasking:
      1. Add 4 drops of Pretreat 3 (included in the CISH Reagent Kit), cover with parafilm and incubate 30 min at 40 oC in the hybridization oven.
    9. Wash slides 3 to 5 times in fresh distilled water.
    10. Add 4 drops of the dapB probe set to those brain slices used to assess background staining.
    11. Add 4 drops of the targeting probe set to those brain slices used to detect the RNA of interest.
      NOTE: A probe set targeting residues 15 - 1,256 of the human ATF4 mRNA is used in this example. OPTIONAL: additional brain slices can be hybridized with a probe set targeting human PPIB and used as positive controls.
    12. Place slides in the slide box and incubate for 2 hrs. at 40 oC in the hybridization oven.
    13. Wash slides twice with 1x wash buffer for 2 min at RT.
    14. Add 4 drops of AMP 1 reagent to each brain slice, cover slides with parafilm, place them in the slide box and incubate at 40 oC for 30 min in the hybridization oven.
    15. Wash slides twice with 1x wash buffer for 2 min at RT.
    16. Add 4 drops of AMP 2 reagent to each brain slice, cover slides with parafilm, place them in the slide box and incubate at 40 oC for 15 min in the hybridization oven.
    17. Wash slides twice with 1x wash buffer for 2 min at RT.
    18. Add 4 drops of AMP 3 reagent to each brain slice, cover slides with parafilm, place them in the slide box and incubate at 40 oC for 30 min in the hybridization oven.
    19. Wash slides twice with 1x wash buffer for 2 min at RT.
    20. Add 4 drops of AMP 4 reagent to each brain slice, cover slides with parafilm, place them in the slide box and incubate at 40 oC for 15 min in the hybridization oven.
    21. Wash slides twice with 1x wash buffer for 2 min at RT.
    22. Add 4 drops of AMP 5 reagent to each brain slice, cover slides with parafilm and incubate 30 min at RT.
    23. Wash slides twice with 1x wash buffer for 2 min at RT.
    24. Add 4 drops of AMP 6 reagent to each brain slice, cover slides with parafilm and incubate 15 min at RT.
    25. Wash slides twice with 1x wash buffer for 2 min at RT.
    26. Mix equal volume of BROWN-1 and BROWN-2 reagents (included in the CISH Reagent Kit), add ~120 μl of solution to each brain slice, cover with parafilm and incubate 10 min at RT.
    27. Wash slides twice with 1x wash buffer for 2 min at RT and rinse once in distilled water.
      NOTE: CRITICAL STEPS: the following steps are critical to obtain an optimal axonal counterstain without masking the presence of RNA granules.
    28. Before performing counterstain check the presence of mRNA of interest under a brightfield microscope. Define reference areas in the brain samples in which to monitor the presence of puncta throughout the counterstain procedure.
    29. Preheat luxol fast blue solution at 60 oC.
    30. Place slides in luxol fast blue and incubate 30 min at 60 oC.
    31. Rinse several times in distilled water.
    32. Dip slides several times in 0.05% lithium carbonate solution to start differentiation.
    33. Dip slides twice in fresh 75% ethanol.
    34. Rinse in distilled water.
    35. Check brain slices under a brightfield microscope. Note that grey and white matter should start to be distinguishable and RNA granules still visible as dark blue/black puncta. Verify that axons start appearing as light blue fibers.
    36. Repeat steps 2.2.28 to 2.2.32. Perform incubations with luxol fast blue solution in 10 - 20 min steps, carefully monitoring the counterstain and the presence of RNA granules. Note: Typically, optimal counterstain is acquired in 60 - 90 min (total incubation time) but time can be shortened if it is suspected that RNA granules might be masked by the luxol fast blue stain (see Figure 2 for examples).
    37. Incubate slides in cresyl violet solution for 10 min at RT.
    38. Rinse slides in distilled water.
    39. Dip slides 5 to 10 times in 70% ethanol.
    40. Dehydrate brain slices through 3 quick changes of 100% ethanol.
    41. In a fume hood, clear brain slices by incubating twice in xylene alternative clearing agent for 2 min and a third time for 5 min at RT.
    42. Mount brain slices in xylene-based permanent mounting medium.
    43. Analyze samples under a brightfield microscope.

Access restricted. Please log in or start a trial to view this content.

Results

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

A brief summary of the procedures described above is shown in Figure 1.

Optimal detection of Atf4 mRNA granules in cholinergic axons using heat-induced unmasking

When assessing axonal localization of mRNAs, it is critical to be able to identify the axons and to be able to visualize low abundant RNAs. The RNA ISH technology described here enables the detection of RNAs at a single-molecule resolution. Standard protocols using this technology ...

Access restricted. Please log in or start a trial to view this content.

Discussion

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

In this report we describe the use of a high-resolution ISH technology in the detection of axonally localized Atf4 mRNA. These and previous published studies show that this technology is compatible with antibody-based protein detection in tissues or even whole embryos33. Importantly, it has been recently used for the detection of Arc mRNA within dendrites of hippocampal neurons34. It can also be combined with histological dyes for tissue staining. Finally, it is suitable for the si...

Access restricted. Please log in or start a trial to view this content.

Disclosures

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

The authors have nothing to disclose.

Acknowledgements

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

This work was supported by the Alzheimer’s Association (NIRG-10-171721; to U.H.), National Institute of Mental Health (MH096702; to U.H.), National Institute of Neurological Disorders and Stroke (NS081333; to C.M.T.), and pilot study awards from the National Institute on Aging-funded Alzheimer’s Disease Research Center at Columbia University (AG008702; to J.B. and Y.Y.J.) that also supports the New York Brain Bank. We thank members of the Hengst laboratory for comments and discussions

Access restricted. Please log in or start a trial to view this content.

Materials

List of materials used in this article
NameCompanyCatalog NumberComments
custom probe targeting residues 20-1,381 of the mouse Atf4 mRNA (NM_009716)Advanced Cell Diagnostics-probe
custom probe targeting residues 15-1,256 of the human ATF4 mRNA (NM_001675.2)Advanced Cell Diagnostics-probe
negative control probe-DapBAdvanced Cell Diagnostics310043probe
positive control probe-mouse Polr2A (optional)Advanced Cell Diagnostics312471probe
positive control probe-human PPIB (optional)Advanced Cell Diagnostics313901probe
RNAscope Fluorescent Multiplex Reagent Kit (for fluorescence detection)Advanced Cell Diagnostics320850in situ hybridization kit
RNAscope 2.0 HD Reagent Kit - BROWN (for chromogenic detection)Advanced Cell Diagnostics310035in situ hybridization kit
Goat polyclonal anti-ChAT antibodyMilliporeAB144P
Luxol Fast Blue-Cresyl Echt Violet Stain KitAmerican MasterTechKTLFB
Clearify clearing agent (xylene substitute)American MasterTechCACLEGAL
ProLong Gold mounting  medium with DAPILife TechnologiesP36935
DPX mounting mediumSigma6522

References

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,
  1. Jung, H., Yoon, B. C., Holt, C. E. Axonal mRNA localization and local protein synthesis in nervous system assembly, maintenance and repair. Nat. Rev. Neurosci. 13, 308-324 (2012).
  2. Campbell, D. S., Holt, C. E. Chemotropic responses of retinal growth cones mediated by rapid local protein synthesis and degradation. Neuron. 32, 1013-1026 (2001).
  3. Wu, K. Y., et al. Local translation of RhoA regulates growth cone collapse. Nature. 436, 1020-1024 (2005).
  4. Piper, M., et al. Signaling mechanisms underlying Slit2-induced collapse of Xenopus retinal growth cones. Neuron. 49, 215-228 (2006).
  5. Yao, J., Sasaki, Y., Wen, Z., Bassell, G. J., Zheng, J. Q. An essential role for β-actin mRNA localization and translation in Ca2+-dependent growth cone guidance. Nat. Neurosci. 9, 1265-1273 (2006).
  6. Hengst, U., Deglincerti, A., Kim, H. J., Jeon, N. L., Jaffrey, S. R. Axonal elongation triggered by stimulus-induced local translation of a polarity complex protein. Nat. Cell Biol. 11, 1024-1030 (2009).
  7. Gracias, N. G., Shirkey-Son, N. J., Hengst, U. Local translation of TC10 is required for membrane expansion during axon outgrowth. Nat. Commun. 5, 3506(2014).
  8. Preitner, N., et al. APC Is an RNA-Binding Protein, and Its Interactome Provides a Link to Neural Development and Microtubule Assembly. Cell. 158, 368-382 (2014).
  9. Brittis, P. A., Lu, Q., Flanagan, J. G. Axonal protein synthesis provides a mechanism for localized regulation at an intermediate target. Cell. 110, 223-235 (2002).
  10. Tcherkezian, J., Brittis, P. A., Thomas, F., Roux, P. P., Flanagan, J. G. Transmembrane receptor DCC associates with protein synthesis machinery and regulates translation. Cell. 141, 632-644 (2010).
  11. Colak, D., Ji, S. J., Porse, B. T., Jaffrey, S. R. Regulation of axon guidance by compartmentalized nonsense-mediated mRNA decay. Cell. 153, 1252-1265 (2013).
  12. Cox, L. J., Hengst, U., Gurskaya, N. G., Lukyanov, K. A., Jaffrey, S. R. Intra-axonal translation and retrograde trafficking of CREB promotes neuronal survival. Nat. Cell Biol. 10, 149-159 (2008).
  13. Ji, S. J., Jaffrey, S. R. Intra-axonal translation of SMAD1/5/8 mediates retrograde regulation of trigeminal ganglia subtype specification. Neuron. 74, 95-107 (2012).
  14. Bassell, G. J., Singer, R. H., Kosik, K. S. Association of poly(A) mRNA with microtubules in cultured neurons. Neuron. 12, 571-582 (1994).
  15. Kleiman, R., Banker, G., Steward, O. Development of subcellular mRNA compartmentation in hippocampal neurons in culture. J. Neurosci. 14, 1130-1140 (1994).
  16. Hengst, U., Jaffrey, S. R. Function and translational regulation of mRNA in developing axons. Semin. Cell Dev. Biol. 18, 209-215 (2007).
  17. Deglincerti, A., Jaffrey, S. R. Insights into the roles of local translation from the axonal transcriptome. Open Biology. 2, 120079(2012).
  18. Baleriola, J., et al. Axonally synthesized ATF4 transmits a neurodegenerative signal across brain regions. Cell. 158, 1159-1172 (2014).
  19. Gumy, L. F., et al. Transcriptome analysis of embryonic and adult sensory axons reveals changes in mRNA repertoire localization. RNA. 17, 85-98 (2011).
  20. Taylor, A. M., et al. Axonal mRNA in uninjured and regenerating cortical mammalian axons. J. Neurosci. 29, 4697-4707 (2009).
  21. Yoon, B. C., et al. Local translation of extranuclear lamin B promotes axon maintenance. Cell. 148, 752-764 (2012).
  22. Dubacq, C., Jamet, S., Trembleau, A. Evidence for developmentally regulated local translation of odorant receptor mRNAs in the axons of olfactory sensory neurons. J. Neurosci. 29, 10184-10190 (2009).
  23. Willis, D. E., et al. Axonal Localization of transgene mRNA in mature PNS and CNS neurons. J. Neurosci. 31, 14481-14487 (2011).
  24. Kar, A. N., et al. Dysregulation of the axonal trafficking of nuclear-encoded mitochondrial mRNA alters neuronal mitochondrial activity and mouse. Dev. Neurobiol. 74, 333-350 (2014).
  25. Levsky, J. M., Singer, R. H. Fluorescence in situ hybridization: past, present and future. J. Cell Sci. 116, 2833-2838 (2003).
  26. Trcek, T., et al. Single-mRNA counting using fluorescent in situ hybridization in budding yeast. Nat. Protoc. 7, 408-419 (2012).
  27. Raj, A., van den Bogaard, P., Rifkin, S. A., van Oudenaarden, A., Tyagi, S. Imaging individual mRNA molecules using multiple singly labeled probes. Nat. Methods. 5, 877-879 (2008).
  28. Wang, F., et al. RNAscope: a novel in situ RNA analysis platform for formalin-fixed, paraffin-embedded tissues. J. Mol. Diagn. 14, 22-29 (2012).
  29. Ge, S., Crooks, G. M., McNamara, G., Wang, X. Fluorescent immunohistochemistry and in situ hybridization analysis of mouse pancreas using low-power antigen-retrieval technique. J. Histochem. Cytochem. 54, 843-847 (2006).
  30. Wang, H., et al. Dual-Color Ultrasensitive Bright-Field RNA In Situ Hybridization with RNAscope. Methods Mol. Biol. 1211, 139-149 (2014).
  31. Kluver, H., Barrera, E. A method for the combined staining of cells and fibers in the nervous system. J. Neuropathol. Exp. Neurol. 12, 400-403 (1953).
  32. Sheehan, D. C., Hrapchak, B. B. Theory and Practice of Histotechnology. , 2nd edn, Battelle Press. (1987).
  33. Gross-Thebing, T., Paksa, A., Raz, E. Simultaneous high-resolution detection of multiple transcripts combined with localization of proteins in whole-mount embryos. BMC Biol. 12, 55(2014).
  34. Farris, S., Lewandowski, G., Cox, C. D., Steward, O. Selective localization of arc mRNA in dendrites involves activity- and translation-dependent mRNA degradation. The Journal of neuroscience : the official journal of the Society for Neuroscience. 34, 4481-4493 (2014).
  35. Wang, F., et al. RNAscope: a novel in situ RNA analysis platform for formalin-fixed, paraffin-embedded tissues. The Journal of molecular diagnostics : JMD. 14, 22-29 (2012).
  36. Sheehan, D. C., Hrapchak, B. B. Theory and practice of histotechnology. , 2d edn, Mosby. (1980).

Access restricted. Please log in or start a trial to view this content.

Reprints and Permissions

Request permission to reuse the text or figures of this JoVE article

Request Permission

Tags

In Situ HybridizationRNAscope TechnologyAxonal mRNA DetectionBrain Section AnalysisFluorescence ImmunohistochemistryAntigen UnmaskingProbe HybridizationSignal AmplificationCholine Acetyltransferase StainingLuxol Fast Blue Counterstain

Related Articles