This work was supported by the Alzheimer&#8217;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&#8217;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

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
<ol>
	<li>Sample preparation for paraformaldehyde-fixed frozen brain slices
	<ol>
		<li>For the following example, prepare slices from fixed frozen mouse brains.
		<ol>
			<li>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).</li>
			<li>Remove brains and post-fix in 4% paraformaldehyde for 24 hrs at 4 oC.</li>
			<li>After fixation, wash samples three times in PBS and cryoprotect in 30% sucrose in PBS (pH 7.4) for 24 hrs at 4 oC.</li>
			<li>Embed brains in OCT freezing compound, serially section at 20 &#181;m on a cryostat and mount on poly-D-lysine treated microscope slides. Keep brain slices at -20 oC until use.</li>
		</ol>
		</li>
		<li>Remove paraformaldehyde-fixed brain slices from freezer and air-dry for 30 min at RT.</li>
		<li>Wash three times with 1x PBS.</li>
		<li>In a fume hood, re-fix brain slices 20 min with 4% paraformaldehyde at RT. 
		&#8203;NOTE: CAUTION: 4% paraformaldehyde is toxic and must be handled and discarded appropriately following safety protocols.</li>
		<li>Wash three times with 1x PBS.</li>
		<li>Dehydrate brain slices.
		<ol>
			<li>Prepare 50%, 75% and 100% ethanol solutions.</li>
			<li>Immerse slides in 50% ethanol for 5 min at RT.</li>
			<li>Immerse slides in 75% ethanol for 5 min at RT.</li>
			<li>Immerse slides in 100% ethanol for 5 min at RT.</li>
			<li>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.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>FISH assay followed by immunohistochemistry using heat-induced antigen/RNA unmasking
	<ol>
		<li>Before starting prepare all required material:
		<ol>
			<li>Heat the hybridization oven to 40 oC and place a tray containing distilled water in the oven to create a humidified environment.</li>
			<li>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.</li>
			<li>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.</li>
			<li>Equilibrate amplification reagents AMP 1-4 (included in the Fluorescent Multiplex Reagent Kit) at RT.</li>
			<li>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.</li>
			<li>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.</li>
		</ol>
		</li>
		<li>Remove slides from 100% ethanol and air-dry for 5 min at RT.</li>
		<li>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.
		<ol>
			<li>Fill up a 1 L beaker with distilled water and place it in the microwave. 
			NOTE: The water will &#8220;buffer&#8221; the heat when performing unmasking.</li>
			<li>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.</li>
			<li>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.</li>
		</ol>
		</li>
		<li>Wash slides three times with 1x PBS.</li>
		<li>Add 2 - 3 drops (~100 &#956;l) 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.</li>
		<li>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.</li>
		<li>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. 
		&#8203;NOTE: OPTIONAL: additional brain slices can be hybridized with a probe set targeting mouse Polr2A and used as positive controls.</li>
		<li>Wash slides twice with 1x wash buffer for 2 min at RT.</li>
		<li>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.</li>
		<li>Wash slides twice with 1x wash buffer for 2 min at RT.</li>
		<li>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.</li>
		<li>Wash slides twice with 1x wash buffer for 2 min at RT.</li>
		<li>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.</li>
		<li>Wash slides twice with 1x wash buffer for 2 min at RT.</li>
		<li>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.</li>
		<li>Wash slides twice with 1x wash buffer for 2 min at RT and twice with 1x PBS.</li>
		<li>Add 100 - 200 &#956;l (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.</li>
		<li>Add 100 - 200 &#956;l 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&#8217;t dry out. If needed, re-apply anti-ChAT antibody solution to brain slices 24 hrs after incubation.</li>
		<li>Wash slides three times in 1x PBS for 5 min at RT.</li>
		<li>Add 100 - 200 &#956;l 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.</li>
		<li>Wash slides three times in 1x PBS for 5 min at RT.</li>
		<li>Wash slides once with distilled water.</li>
		<li>Mount slides with DAPI-containing mounting medium.</li>
		<li>Visualize brain slices under a fluorescence microscope.</li>
	</ol>
	</li>
</ol>
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
<ol>
	<li>Sample preparation for formalin-fixed paraffin-embedded human brain samples
	<ol>
		<li>Bake slices in a dry oven at 60 oC for 1 hr.</li>
		<li>Deparaffinize brain slices in a fume hood. 
		&#160;NOTE: CAUTION: reagents are toxic and must be handled and discarded following the appropriate security guidelines
		<ol>
			<li>Immerse slides in xylene alternative clearing agent twice for 10 min.</li>
			<li>Immerse slides in 100% ethanol twice for 1 min.</li>
			<li>Air-dry slices 5 min at RT. Note: Alternatively samples might be dried at RT O/N but must be used within 24 hrs.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Diaminobenzidine (DAB)-based chromogenic ISH assay followed by luxol fast blue and cresyl violet stain using heat-induced and protease-induced RNA unmasking
	<ol>
		<li>Before starting prepare required materials:
		<ol>
			<li>Heat the hybridization oven to 40 oC and place a tray containing distilled water to create a humidified environment.</li>
			<li>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.</li>
			<li>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).&#160;Heat solution to 100 oC and keep it boiling no longer than 30 min before submerging samples.</li>
			<li>Heat negative and positive probes 10 min at 40 oC and cool down at RT.</li>
			<li>Equilibrate amplification reagents AMP 1-6 (included in the CISH Reagent Kit) at RT.</li>
			<li>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.</li>
		</ol>
		</li>
		<li>Add ~4 drops (~120 &#956;l) of Pretreat 1 to each brain slice, cover with parafilm and incubate at RT for 10 min.</li>
		<li>Wash 3 to 5 times in fresh distilled water.</li>
		<li>Perform heat-induced unmasking:
		<ol>
			<li>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&#160;1.2.3).&#160;</li>
		</ol>
		</li>
		<li>Immediately transfer slides to distilled water and wash 3 to 5 times.</li>
		<li>Wash slides 3 to 5 times in fresh 100% ethanol.</li>
		<li>Air-dry slices 5 min at RT. Note: Alternatively slices can be dried O/N.</li>
		<li>Perform protease-induced unmasking:
		<ol>
			<li>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.</li>
		</ol>
		</li>
		<li>Wash slides 3 to 5 times in fresh distilled water.</li>
		<li>Add 4 drops of the dapB probe set to those brain slices used to assess background staining.</li>
		<li>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.</li>
		<li>Place slides in the slide box and incubate for 2 hrs. at 40 oC in the hybridization oven.</li>
		<li>Wash slides twice with 1x wash buffer for 2 min at RT.</li>
		<li>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.</li>
		<li>Wash slides twice with 1x wash buffer for 2 min at RT.</li>
		<li>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.</li>
		<li>Wash slides twice with 1x wash buffer for 2 min at RT.</li>
		<li>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.</li>
		<li>Wash slides twice with 1x wash buffer for 2 min at RT.</li>
		<li>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.</li>
		<li>Wash slides twice with 1x wash buffer for 2 min at RT.</li>
		<li>Add 4 drops of AMP 5 reagent to each brain slice, cover slides with parafilm and incubate 30 min at RT.</li>
		<li>Wash slides twice with 1x wash buffer for 2 min at RT.</li>
		<li>Add 4 drops of AMP 6 reagent to each brain slice, cover slides with parafilm and incubate 15 min at RT.</li>
		<li>Wash slides twice with 1x wash buffer for 2 min at RT.</li>
		<li>Mix equal volume of BROWN-1 and BROWN-2 reagents (included in the CISH Reagent Kit), add ~120 &#956;l of solution to each brain slice, cover with parafilm and incubate 10 min at RT.</li>
		<li>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.</li>
		<li>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.</li>
		<li>Preheat luxol fast blue solution at 60 oC.</li>
		<li>Place slides in luxol fast blue and incubate 30 min at 60 oC.</li>
		<li>Rinse several times in distilled water.</li>
		<li>Dip slides several times in 0.05% lithium carbonate solution to start differentiation.</li>
		<li>Dip slides twice in fresh 75% ethanol.</li>
		<li>Rinse in distilled water.</li>
		<li>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.</li>
		<li>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).</li>
		<li>Incubate slides in cresyl violet solution for 10 min at RT.</li>
		<li>Rinse slides in distilled water.</li>
		<li>Dip slides 5 to 10 times in 70% ethanol.</li>
		<li>Dehydrate brain slices through 3 quick changes of 100% ethanol.</li>
		<li>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.</li>
		<li>Mount brain slices in xylene-based permanent mounting medium.</li>
		<li>Analyze samples under a brightfield microscope.</li>
	</ol>
	</li>
</ol>

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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+in+situ+hybridization:+past,+present+and+future.">Fluorescence in situ hybridization: past, present and future.</a> J. Cell Sci. 116, 2833-2838 (2003).</li><li>Trcek, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-mRNA+counting+using+fluorescent+in+situ+hybridization+in+budding+yeast.">Single-mRNA counting using fluorescent in situ hybridization in budding yeast.</a> Nat. Protoc. 7, 408-419 (2012).</li><li>Raj, A., van den Bogaard, P., Rifkin, S. A., van Oudenaarden, A., Tyagi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+individual+mRNA+molecules+using+multiple+singly+labeled+probes.">Imaging individual mRNA molecules using multiple singly labeled probes.</a> Nat. Methods. 5, 877-879 (2008).</li><li>Wang, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNAscope:+a+novel+in+situ+RNA+analysis+platform+for+formalin-fixed,+paraffin-embedded+tissues.">RNAscope: a novel in situ RNA analysis platform for formalin-fixed, paraffin-embedded tissues.</a> J. Mol. Diagn. 14, 22-29 (2012).</li><li>Ge, S., Crooks, G. M., McNamara, G., Wang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+immunohistochemistry+and+in+situ+hybridization+analysis+of+mouse+pancreas+using+low-power+antigen-retrieval+technique.">Fluorescent immunohistochemistry and in situ hybridization analysis of mouse pancreas using low-power antigen-retrieval technique.</a> J. Histochem. Cytochem. 54, 843-847 (2006).</li><li>Wang, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dual-Color+Ultrasensitive+Bright-Field+RNA+In+Situ+Hybridization+with+RNAscope.">Dual-Color Ultrasensitive Bright-Field RNA In Situ Hybridization with RNAscope.</a> Methods Mol. Biol. 1211, 139-149 (2014).</li><li>Kluver, H., Barrera, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+the+combined+staining+of+cells+and+fibers+in+the+nervous+system.">A method for the combined staining of cells and fibers in the nervous system.</a> J. Neuropathol. Exp. Neurol. 12, 400-403 (1953).</li><li>Sheehan, D. C., Hrapchak, B. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Theory and Practice of Histotechnology. , (1987).</li><li>Gross-Thebing, T., Paksa, A., Raz, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+high-resolution+detection+of+multiple+transcripts+combined+with+localization+of+proteins+in+whole-mount+embryos.">Simultaneous high-resolution detection of multiple transcripts combined with localization of proteins in whole-mount embryos.</a> BMC Biol. 12, 55 (2014).</li><li>Farris, S., Lewandowski, G., Cox, C. D., Steward, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+localization+of+arc+mRNA+in+dendrites+involves+activity-+and+translation-dependent+mRNA+degradation.">Selective localization of arc mRNA in dendrites involves activity- and translation-dependent mRNA degradation.</a> The Journal of neuroscience : the official journal of the Society for Neuroscience. 34, 4481-4493 (2014).</li><li>Wang, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNAscope:+a+novel+in+situ+RNA+analysis+platform+for+formalin-fixed,+paraffin-embedded+tissues.">RNAscope: a novel in situ RNA analysis platform for formalin-fixed, paraffin-embedded tissues.</a> The Journal of molecular diagnostics : JMD. 14, 22-29 (2012).</li><li>Sheehan, D. C., Hrapchak, B. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Theory and practice of histotechnology. , (1980).</li></ol>

The authors have nothing to disclose.

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...

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&#8217;s disease peptide A&#946;1-42, and local translation of the transcription factor ATF4 is required to propagate the neurodegenerative effects of A&#946;1-42 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&#8217; UTR of &#946;-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 A&#946;1-42-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.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>custom probe targeting residues 20-1381 of the mouse Atf4 mRNA (NM_009716)</td>
			<td>Advanced Cell Diagnostics</td>
			<td>-</td>
			<td>probe</td>
		</tr>
		<tr>
			<td>custom probe targeting residues 15-1256 of the human ATF4 mRNA (NM_001675.2)</td>
			<td>Advanced Cell Diagnostics</td>
			<td>-</td>
			<td>probe</td>
		</tr>
		<tr>
			<td>negative control probe-DapB</td>
			<td>Advanced Cell Diagnostics</td>
			<td align="right">310043</td>
			<td>probe</td>
		</tr>
		<tr>
			<td>positive control probe-mouse Polr2A (optional)</td>
			<td>Advanced Cell Diagnostics</td>
			<td align="right">312471</td>
			<td>probe</td>
		</tr>
		<tr>
			<td>positive control probe-human PPIB (optional)</td>
			<td>Advanced Cell Diagnostics</td>
			<td align="right">313901</td>
			<td>probe</td>
		</tr>
		<tr>
			<td>RNAscope Fluorescent Multiplex Reagent Kit (for fluorescence detection)</td>
			<td>Advanced Cell Diagnostics</td>
			<td align="right">320850</td>
			<td>In situ hybridization kit</td>
		</tr>
		<tr>
			<td>RNAscope 2.0 HD Reagent Kit - BROWN (for chromogenic detection)</td>
			<td>Advanced Cell Diagnostics</td>
			<td align="right">310035</td>
			<td>In situ hybridization kit</td>
		</tr>
		<tr>
			<td>Goat polyclonal anti-ChAT antibody</td>
			<td>Millipore</td>
			<td>AB144P</td>
			<td></td>
		</tr>
		<tr>
			<td>Luxol Fast Blue-Cresyl Echt Violet Stain Kit</td>
			<td>American MasterTech</td>
			<td>KTLFB</td>
			<td></td>
		</tr>
		<tr>
			<td>Clearify clearing agent (xylene substitute)</td>
			<td>American MasterTech</td>
			<td>CACLEGAL</td>
			<td></td>
		</tr>
		<tr>
			<td>ProLong Gold mounting&#160; medium with DAPI</td>
			<td>Life Technologies</td>
			<td>P36935</td>
			<td></td>
		</tr>
		<tr>
			<td>DPX mounting medium</td>
			<td>Sigma</td>
			<td align="right">6522</td>
			<td></td>
		</tr>
	</tbody>
</table>

detection of axonally localized mrnas in brain sections using high-resolution in situ hybridization

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.

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 ...

Watch this Scientific Journal Video about Detection of Axonally Localized mRNAs in Brain Sections Using High-Resolution In Situ Hybridization at JoVE.com

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

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.

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

mRNAs are frequently localized to vertebrate axons and their local translation is required for axon pathfinding or branching during development and for ...

detection-axonally-localized-mrnas-brain-sections-using-high

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Neuroscience

11.7K Views.  Columbia University. 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.

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

Double Fluorescence in situ Hybridization in Fresh Brain Sections

Here we describe a modified version of a double fluorescence in situ hybridization (dFISH) method optimized for detecting two mRNAs of interest in fresh frozen brain sections. Our group has successfully used this approach to study gene co-regulation. More specifically, we have used this dFISH method to explore the anatomical organization, neurochemical properties, and the impact of sensory experience in central sensory circuits, at single cell resolution. This protocol has been validated in brain tissue from mice, rats and songbirds but is expected to be easily adaptable to other vertebrate species, as well as to an array of non-neural tissues. In this film we provide a detailed demonstration of the main steps of this procedure.

Double Fluorescence in situ Hybridization in Fresh Brain Sections

This protocol involves a non-radioactive in-situ hybridization procedure that enables the simultaneous identification of two transcript species, at a single cell resolution, in thin sections of the vertebrate brain.

Here we describe a modified version of a double fluorescence in situ hybridization (dFISH) method optimized for detecting two mRNAs of interest in fresh ...

Combining Lipophilic dye, in situ Hybridization, Immunohistochemistry, and Histology

Going beyond single gene function to cut deeper into gene regulatory networks requires multiple mutations combined in a single animal. Such analysis of two or more genes needs to be complemented with in situ hybridization of other genes, or immunohistochemistry of their proteins, both in whole mounted developing organs or sections for detailed resolution of the cellular and tissue expression alterations. Combining multiple gene alterations requires the use of cre or flipase to conditionally delete genes and avoid embryonic lethality. Required breeding schemes dramatically enhance effort and cost proportional to the number of genes mutated, with an outcome of very few animals with the full repertoire of genetic modifications desired. Amortizing the vast amount of effort and time to obtain these few precious specimens that are carrying multiple mutations necessitates tissue optimization. Moreover, investigating a single animal with multiple techniques makes it easier to correlate gene deletion defects with expression profiles. We have developed a technique to obtain a more thorough analysis of a given animal; with the ability to analyze several different histologically recognizable structures as well as gene and protein expression all from the same specimen in both whole mounted organs and sections. Although mice have been utilized to demonstrate the effectiveness of this technique it can be applied to a wide array of animals. To do this we combine lipophilic dye tracing, whole mount in situ hybridization, immunohistochemistry, and histology to extract the maximal possible amount of data.

Combining Lipophilic dye, in situ Hybridization, Immunohistochemistry, and Histology

A combination of different techniques to maximize data collection from mouse tissue is presented.

Going beyond single gene function to cut deeper into gene regulatory networks requires multiple mutations combined in a single animal.  Such analysis of ...

A Rapid Approach to High-Resolution Fluorescence Imaging in Semi-Thick Brain Slices

A fundamental goal to both basic and clinical neuroscience is to better understand the identities, molecular makeup, and patterns of connectivity that are characteristic to neurons in both normal and diseased brain. Towards this, a great deal of effort has been placed on building high-resolution neuroanatomical maps1-3. With the expansion of molecular genetics and advances in light microscopy has come the ability to query not only neuronal morphologies, but also the molecular and cellular makeup of individual neurons and their associated networks4. Major advances in the ability to mark and manipulate neurons through transgenic and gene targeting technologies in the rodent now allow investigators to 'program' neuronal subsets at will5-6. Arguably, one of the most influential contributions to contemporary neuroscience has been the discovery and cloning of genes encoding fluorescent proteins (FPs) in marine invertebrates7-8, alongside their subsequent engineering to yield an ever-expanding toolbox of vital reporters9. Exploiting cell type-specific promoter activity to drive targeted FP expression in discrete neuronal populations now affords neuroanatomical investigation with genetic precision.
Engineering FP expression in neurons has vastly improved our understanding of brain structure and function. However, imaging individual neurons and their associated networks in deep brain tissues, or in three dimensions, has remained a challenge. Due to high lipid content, nervous tissue is rather opaque and exhibits auto fluorescence. These inherent biophysical properties make it difficult to visualize and image fluorescently labelled neurons at high resolution using standard epifluorescent or confocal microscopy beyond depths of tens of microns. To circumvent this challenge investigators often employ serial thin-section imaging and reconstruction methods10, or 2-photon laser scanning microscopy11. Current drawbacks to these approaches are the associated labor-intensive tissue preparation, or cost-prohibitive instrumentation respectively.
Here, we present a relatively rapid and simple method to visualize fluorescently labelled cells in fixed semi-thick mouse brain slices by optical clearing and imaging. In the attached protocol we describe the methods of: 1) fixing brain tissue in situ via intracardial perfusion, 2) dissection and removal of whole brain, 3) stationary brain embedding in agarose, 4) precision semi-thick slice preparation using new vibratome instrumentation, 5) clearing brain tissue through a glycerol gradient, and 6) mounting on glass slides for light microscopy and z-stack reconstruction (Figure 1).
For preparing brain slices we implemented a relatively new piece of instrumentation called the 'Compresstome' VF-200 (http://www.precisionary.com/products_vf200.html). This instrument is a semi-automated microtome equipped with a motorized advance and blade vibration system with features similar in function to other vibratomes. Unlike other vibratomes, the tissue to be sliced is mounted in an agarose plug within a stainless steel cylinder. The tissue is extruded at desired thicknesses from the cylinder, and cut by the forward advancing vibrating blade. The agarose plug/cylinder system allows for reproducible tissue mounting, alignment, and precision cutting. In our hands, the 'Compresstome' yields high quality tissue slices for electrophysiology, immunohistochemistry, and direct fixed-tissue mounting and imaging. Combined with optical clearing, here we demonstrate the preparation of semi-thick fixed brain slices for high-resolution fluorescent imaging.

Here we describe a rapid and simple method to image fluorescently labeled cells in semi-thick brain slices. By fixing, slicing, and optically clearing brain tissue we describe how standard epifluorescent or confocal imaging can be used to visualize individual cells and neuronal networks within intact nervous tissue.

A fundamental goal to both basic and clinical neuroscience is to better understand the identities, molecular makeup, and patterns of connectivity that are ...

Detection of the Genome and Transcripts of a Persistent DNA Virus in Neuronal Tissues by Fluorescent In situ Hybridization Combined with Immunostaining

Single cell codetection of a gene, its RNA product and cellular regulatory proteins is critical to study gene expression regulation. This is a challenge in the field of virology; in particular for nuclear-replicating persistent DNA viruses that involve animal models for their study. Herpes simplex virus type 1 (HSV-1) establishes a life-long latent infection in peripheral neurons. Latent virus serves as reservoir, from which it reactivates and induces a new herpetic episode. The cell biology of HSV-1 latency remains poorly understood, in part due to the lack of methods to detect HSV-1 genomes in situ in animal models. We describe a DNA-fluorescent in situ hybridization (FISH) approach efficiently detecting low-copy viral genomes within sections of neuronal tissues from infected animal models. The method relies on heat-based antigen unmasking, and directly labeled home-made DNA probes, or commercially available probes. We developed a triple staining approach, combining DNA-FISH with RNA-FISH&#160;and immunofluorescence, using peroxidase based signal amplification to accommodate each staining requirement. A major improvement is the ability to obtain, within 10 &#181;m tissue sections, low-background signals that can be imaged at high resolution by confocal microscopy and wide-field conventional epifluorescence. Additionally, the triple staining worked with a wide range of antibodies directed against cellular and viral proteins. The complete protocol takes 2.5 days to accommodate antibody and probe penetration within the tissue.

Detection of the Genome and Transcripts of a Persistent DNA Virus in Neuronal Tissues by Fluorescent In situ Hybridization Combined with Immunostaining

We established a fluorescent in situ hybridization protocol for the detection of a persistent DNA virus genome within tissue sections of animal models. This protocol enables studying infection process by codetection of the viral genome, its RNA products, and viral or cellular proteins within single cells.

Single cell codetection of a gene, its RNA product and cellular regulatory proteins is critical to study gene expression regulation. This is a challenge ...

Complete Spinal Cord Injury and Brain Dissection Protocol for Subsequent Wholemount In Situ Hybridization in Larval Sea Lamprey

After a complete spinal cord injury, sea lampreys at first are paralyzed below the level of transection. However, they recover locomotion after several weeks, and this is accompanied by short distance regeneration (a few mm) of propriospinal axons and spinal-projecting axons from the brainstem. Among the 36 large identifiable spinal-projecting neurons, some are good regenerators and others are bad regenerators. These neurons can most easily be identified in wholemount CNS preparations. In order to understand the neuron-intrinsic mechanisms that favor or inhibit axon regeneration after injury in the vertebrates CNS, we determine differences in gene expression between the good and bad regenerators, and how expression is influenced by spinal cord transection. This paper illustrates the techniques for housing larval and recently transformed adult sea lampreys in fresh water tanks, producing complete spinal cord transections under microscopic vision, and preparing brain and spinal cord wholemounts for in situ hybridization. Briefly, animals are kept at 16&#160;&#176;C and anesthetized in 1% Benzocaine in lamprey Ringer. The spinal cord is transected with iridectomy scissors via a dorsal approach and the animal is allowed to recover in fresh water tanks at 23 &#176;C. For in situ hybridization, animals are reanesthetized and the brain and cord removed via a dorsal approach.

Complete Spinal Cord Injury and Brain Dissection Protocol for Subsequent Wholemount In Situ Hybridization in Larval Sea Lamprey

Lampreys recover locomotion after a complete spinal cord injury. However, some spinal-projecting neurons are good regenerators and others are not. This paper illustrates the techniques for housing sea lamprey larvae (and recently transformed adults), producing complete spinal cord transections and preparing wholemount brains and spinal cords for in situ hybridization.

After a complete spinal cord injury, sea lampreys at first are paralyzed below the level of transection. However, they recover locomotion after several ...

High-resolution In Vivo Manual Segmentation Protocol for Human Hippocampal Subfields Using 3T Magnetic Resonance Imaging

The human hippocampus has been broadly studied in the context of memory and normal brain function and its role in different neuropsychiatric disorders has been heavily studied. While many imaging studies treat the hippocampus as a single unitary neuroanatomical structure, it is, in fact, composed of several subfields that have a complex three-dimensional geometry. As such, it is known that these subfields perform specialized functions and are differentially affected through the course of different disease states. Magnetic resonance (MR) imaging can be used as a powerful tool to interrogate the morphology of the hippocampus and its subfields. Many groups use advanced imaging software and hardware (&#62;3T) to image the subfields; however this type of technology may not be readily available in most research and clinical imaging centers. To address this need, this manuscript provides a detailed step-by-step protocol for segmenting the full anterior-posterior length of the hippocampus and its subfields: cornu ammonis (CA) 1, CA2/CA3, CA4/dentate gyrus (DG), strata radiatum/lacunosum/moleculare (SR/SL/SM), and subiculum. This protocol has been applied to five subjects (3F, 2M; age 29-57, avg. 37). Protocol reliability is assessed by resegmenting either the right or left hippocampus of each subject and computing the overlap using the Dice&#39;s kappa metric. Mean Dice&#39;s kappa (range) across the five subjects are: whole hippocampus, 0.91 (0.90-0.92); CA1, 0.78 (0.77-0.79); CA2/CA3, 0.64 (0.56-0.73); CA4/dentate gyrus, 0.83 (0.81-0.85); strata radiatum/lacunosum/moleculare, 0.71 (0.68-0.73); and subiculum 0.75 (0.72-0.78). The segmentation protocol presented here provides other laboratories with a reliable method to study the hippocampus and hippocampal subfields in vivo using commonly available MR tools.

High-resolution In Vivo Manual Segmentation Protocol for Human Hippocampal Subfields Using 3T Magnetic Resonance Imaging

The goal of this manuscript is to study the hippocampus and hippocampal subfields using MRI. The manuscript describes a protocol for segmenting the hippocampus and five hippocampal substructures: cornu ammonis (CA) 1, CA2/CA3, CA4/dentate gyrus, strata radiatum/lacunosum/moleculare, and subiculum.

The human hippocampus has been broadly studied in the context of memory and normal brain function and its role in different neuropsychiatric disorders has ...

Rapid In Situ Hybridization using Oligonucleotide Probes on Paraformaldehyde-prefixed Brain of Rats with Serotonin Syndrome

3,4-Methylenedioxymethamphetamine (MDMA; ecstasy) toxicity may cause region-specific changes in serotonergic mRNA expression due to acute serotonin (5-hydroxytryptamine; 5-HT) syndrome. This hypothesis can be tested using in situ hybridization to detect the serotonin 5-HT2A receptor gene htr2a. In the past, such procedures, utilizing radioactive riboprobe, were difficult because of the complicated workflow that needs several days to perform and the added difficulty that the technique required the use of fresh frozen tissues maintained in an RNase-free environment. Recently, the development of short oligonucleotide probes has simplified in situ hybridization procedures and allowed the use of paraformaldehyde-prefixed brain sections, which are more widely available in laboratories. Here, we describe a detailed protocol using non-radioactive oligonucleotide probes on the prefixed brain tissues. Hybridization probes used for this study include dapB (a bacterial gene coding for dihydrodipicolinate reductase), ppiB (a housekeeping gene coding for peptidylprolyl isomerase B), and htr2a (a serotonin gene coding for 5-HT2A receptors). This method is relatively simply, cheap, reproducible and requires less than two days to complete.

Rapid In Situ Hybridization using Oligonucleotide Probes on Paraformaldehyde-prefixed Brain of Rats with Serotonin Syndrome

This protocol describes a rapid and simplified in situ hybridization method ideal forparaformaldehyde-prefixed brain, thus reducing the need for prolonged complex steps while using fresh frozen tissues. The method is validated using the identification of the serotonin 5-HT2A receptor gene htr2a in rats.

3,4-Methylenedioxymethamphetamine (MDMA; ecstasy) toxicity may cause region-specific changes in serotonergic mRNA expression due to acute serotonin ...

Combining Double Fluorescence In Situ Hybridization with Immunolabelling for Detection of the Expression of Three Genes in Mouse Brain Sections

Detection of gene expression in different types of brain cells e.g., neurons, astrocytes, oligodendrocytes, oligodendrocyte precursors and microglia, can be hampered by the lack of specific primary or secondary antibodies for immunostaining. Here we describe a protocol to detect the expression of three different genes in the same brain section using double fluorescence in situ hybridization with two gene-specific probes followed by immunostaining with an antibody of high specificity directed against the protein encoded by a third gene. The Aspartoacyclase (ASPA) gene, mutations of which can lead to a rare human white matter disease - Canavan disease - is thought to be expressed in oligodendrocytes and microglia but not in astrocytes and neurons. However, the precise expression pattern of ASPA in the brain has yet to be established. This protocol has allowed us to determine that ASPA is expressed in a subset of mature oligodendrocytes and it can be generally applied to a wide range of gene expression pattern studies.

Combining Double Fluorescence In Situ Hybridization with Immunolabelling for Detection of the Expression of Three Genes in Mouse Brain Sections

Localizing gene expression to specific cell types can be challenging due to the lack of specific antibodies. Here we describe a protocol for simultaneous triple detection of gene expression by combining double fluorescence RNA in situ hybridization with immunostaining.

Detection of gene expression in different types of brain cells e.g., neurons, astrocytes, oligodendrocytes, oligodendrocyte precursors and microglia, can ...

Identification of Dopamine D1-Alpha Receptor Within Rodent Nucleus Accumbens by an Innovative RNA In Situ Detection Technology

In the central nervous system, the D1-alpha subtype receptor (Drd1&#945;) is the most abundant dopamine (DA) receptor, which plays a vital role in regulating neuronal growth and development. However, the mechanisms underlying Drd1&#945; receptor abnormalities mediating behavioral responses and modulating working memory function are still unclear. Using a novel RNA in situ hybridization assay, the current study identified dopamine Drd1&#945; receptor and tyrosine hydroxylase (TH) RNA expression from DA-related circuitry in the nucleus accumbens (NAc) area and substantia nigra region (SNR), respectively. Drd1&#945; expression in the NAc shows a &#34;discrete dot&#34; staining pattern. Clear sex differences in Drd1&#945; expression were observed. In contrast, TH shows a &#34;clustered&#34; staining pattern. Regarding TH expression, female rats displayed a higher signal expression per cell relative to male animals. The methods presented here provide a novel in situ hybridization technique for investigating changes in dopamine system dysfunction during the progression of central nervous system diseases.

Identification of Dopamine D1-Alpha Receptor Within Rodent Nucleus Accumbens by an Innovative RNA In Situ Detection Technology

Identification of dopamine D1-alpha receptor in the nucleus accumbens is critical for clarifying D1 receptor dysfunction during a central nervous system disease. We performed a novel RNA in situ hybridization assay to visualize single RNA molecules in a specific brain area.

In the central nervous system, the D1-alpha subtype receptor (Drd1&#945;) is the most abundant dopamine (DA) receptor, which plays a vital role in ...

High-Resolution 3D Imaging of Rabies Virus Infection in Solvent-Cleared Brain Tissue

The visualization of infection processes in tissues and organs by immunolabeling is a key method in modern infection biology. The ability to observe and study the distribution, tropism, and abundance of pathogens inside of organ tissues provides pivotal data on disease development and progression. Using conventional microscopy methods, immunolabeling is mostly restricted to thin sections obtained from paraffin-embedded or frozen samples. However, the limited 2D image plane of these thin sections may lead to the loss of crucial information on the complex structure of an infected organ and the cellular context of the infection. Modern multicolor, immunostaining-compatible tissue clearing techniques now provide a relatively fast and inexpensive way to study high-volume 3D image stacks of virus-infected organ tissue. By exposing the tissue to organic solvents, it becomes optically transparent. This matches the sample&#8217;s refractive indices and eventually leads to a significant reduction of light scattering. Thus, in combination with long free working distance objectives, large tissue sections up to 1 mm in size can be imaged by conventional confocal laser scanning microscopy (CLSM) at high resolution. Here, we describe a protocol to apply deep-tissue imaging after tissue clearing to visualize rabies virus distribution in infected brains in order to study topics like virus pathogenesis, spread, tropism, and neuroinvasion.

Novel, immunostaining-compatible tissue clearing techniques like the ultimate 3D imaging of solvent-cleared organs allow the 3D visualization of rabies virus brain infection and its complex cellular environment. Thick, antibody-labeled brain tissue slices are made optically transparent to increase imaging depth and to enable 3D analysis by confocal laser scanning microscopy.

The visualization of infection processes in tissues and organs by immunolabeling is a key method in modern infection biology. The ability to observe and ...