This work was supported by the Neuroanatomy/Histology/Brain Injection Core funded by NIH grant #5P01DK119130-02. The authors would like to acknowledge the assistance of the UT Southwestern Live Cell Imaging Facility (headed by Dr. Phelps) and its staff (Abhijit Bugde and Marcel Mettlen), supported in part by the NIH Grant #1S10OD021684-01, a Shared Resource of the Harold C. Simmons Cancer Center, supported in part by an NCI Cancer Center Support Grant, P30 CA142543.

NOTE: Mice used in this study were wild-type males on a pure C57BL/6J background. A total of 4 mice were used for multiplex ISH. All mice were approximately 8 weeks old at the time of sacrifice. One male mouse (approximately one-year-old) was also used to demonstrate endogenous fluorescence associated with aging. Animals were housed in ventilated cages within a barrier facility with ad libitum access to food and water. The UT Southwestern Medical Center Institutional Animal Care and Use Committee reviewed and approved the procedures described below. Details about reagents and tools can be found in the Table of Materials.
1. Sample collection
<ol>
	<li>On the day of sacrifice, administer an overdose of chloral hydrate (500 mg/kg, i.p.) to the mice. Perfuse the deeply anesthetized mice transcardially using a peristaltic pump with 5 mL of 1x phosphate-buffered saline (PBS) followed by 50 mL of 10% formalin at room temperature. 
	NOTE: This is done in a fume hood to prevent inhalation of formalin.</li>
	<li>As the nodose, petrosal, and jugular ganglia are fused to form a single ganglionic mass in the mouse15, carefully collect the entire vagal ganglionic mass from each side (left and right) with the help of a dissecting scope and fine spring scissors and forceps (see the Table of Materials).</li>
	<li>When decapitating the mouse with a pair of large scissors (see the Table of Materials), avoid crushing the brainstem.</li>
	<li>Following a dissection approach described before in the rat40, remove the sternohyoid and omohyoid muscles lateral to the trachea with small forceps (Table of Materials) to expose the occipital bone. Clear the whole region of muscle, adipose tissue, and conjunctive tissue until the carotid artery, vagus nerve, the hypoglossal nerve, and foramen magnum are visible. Cut the entire hypoglossal nerve with small spring scissors (Table of Materials).</li>
	<li>Look for the nodose ganglion as a translucent mass close to the foramen magnum15. Using small scissors (Table of Materials), carefully break the occipital bone to further expose the jugular ganglion. Look for black melanocytes at the surface of the jugular as a visual landmark.</li>
	<li>Cut nerve attachments between the vagal ganglionic mass and extract the whole ganglionic mass by gently pulling on the peripheral end of the vagus nerve while simultaneously cutting the jugular ganglion towards the brainstem with small spring scissors (Table of Materials).</li>
	<li>Remove the conjunctive tissue and fat sticking to the vagus nerve and ganglionic mass as much as possible.</li>
	<li>As it is easy to lose one ganglion during the transfer from one tube to the other, keep ~0.5 cm of the vagus nerve to facilitate handling and localization of samples.</li>
	<li>Place ganglia with the help of fine forceps in 1.5 mL microcentrifuge tubes and incubate at 4 &#176;C in formalin for 24 h and with 30% sucrose for another 24 h.</li>
	<li>Position ganglia inside a small drop (~4 mm &#216;) of optimum cutting temperature (OCT) medium on a piece of aluminum foil. Next, freeze the sample on a bed of dry ice.</li>
	<li>Cut the samples with a cryostat at -20 &#176;C into sections of 14 &#181;m thickness and collect on glass microscope slides as 3 series 42 &#181;m apart. 
	NOTE: Avoid placing sections close to the edges of the slide. To save reagents, keep the tissue sections as tightly packed as possible but without overlapping.</li>
	<li>Store the samples in a -80 &#176;C freezer until needed for ISH for at least 6 months. 
	&#8203;NOTE: Refer to Figure 1 for troubleshooting for endogenous fluorescence.</li>
</ol>
2. Pretreatment and ISH
<ol>
	<li>Before the day of the experiments, autoclave laboratory glassware to be used for ISH, including staining dishes and washing buffer jars. 
	NOTE: While working under RNase-free conditions is not critical, wear gloves at all times. Keep RNase decontamination solution bottles (Table of Materials) within reach in case contamination is suspected.</li>
	<li>On the first day, bring the slides to room temperature on a bench specifically dedicated to ISH. Rinse the slides in 1x PBS and bake them for 30 mins at 60 &#176;C in a baking oven (Table of Materials).</li>
	<li>Post-fix the slides in 4% formalin for 15 min at 4 &#176;C.</li>
	<li>Bring the reagents in the multiplex kit (Table of Materials) to room temperature.</li>
	<li>Dehydrate the slides in 50%, 70%, and 100% ethanol for 5 min each. Apply H2O2 solution to the samples for 10 min and then rinse in double-distilled water (ddH2O).</li>
	<li>Treat the samples with the target retrieval reagent for 5 min, rinse in ddH2O followed by 100% ethanol, and air-dry. Using a hydrophobic pen, create a barrier around the samples. 
	NOTE: Do not apply the hydrophobic barrier too close to the tissue sections.</li>
	<li>Apply protease for 30 min at 40 &#176;C in a hybridization oven (Table of Materials).</li>
	<li>In the meantime, prewarm the probes at 40 &#176;C and cool them at room temperature before use. Incubate the slides with the desired combination of target probes (Cck1r-C3, Ghsr-C1, Phox2b-C2; or Cck1r-C3, Ghsr-C1, Prdm12-C2) for 2 h at 40 &#176;C in a hybridization oven.</li>
	<li>Incubate negative control tissue with the dihydrodipicolinate reductase (DapB) target C1 probe for 2 h at 40 &#176;C in a hybridization oven.</li>
	<li>Rinse the slides in wash buffer and store overnight in 5x saline sodium citrate (SSC) at room temperature. For convenience, split the experiment into two days. On the following day, rinse the slides with wash buffer and incubate them with amplification reagents listed below (see the user manual in Table of Materials).</li>
	<li>Apply AMP1 (30 min at 40 &#176;C), AMP2 (30 min at 40 &#176;C), and AMP 3 (15 min at 40 &#176;C). Rinse in wash buffer between each step.</li>
	<li>Dissolve each Opal dye (Table of Materials) in dimethyl sulfoxide and store the solutions at 4 &#176;C until needed.</li>
	<li>For channel 1, incubate the slides with HRP-C1 (15 min at 40 &#176;C) and Opal 520 (green color; dilution 1/1,500; 30 mins at 40&#176;C). Rinse in wash buffer between each step.</li>
	<li>For channel 2, incubate the slides with HRP-C2 (15 min at 40 &#176;C) and Opal 570 (yellow color; dilution 1/1,500; for 30 min at 40 &#176;C). Rinse in wash buffer between each step.</li>
	<li>For channel 3, incubate the slides with HRP-C3 (15 min at 40 &#176;C) and Opal 690 (far red color; dilution 1/1,500; for 30 min at 40 &#176;C). Rinse in wash buffer between each step.</li>
	<li>Wash the slides and expose them to 4&#8242;,6-diamidino-2-phenylindole (DAPI) for 30 s.</li>
	<li>Immediately apply mounting medium on each slide and place a coverslip over the tissue section.</li>
	<li>Keep the tissues horizontal in a slide holder at 4 &#176;C until imaging.</li>
</ol>
3. Microscopy and data analysis
NOTE: Multiplex ISH data can be imaged with a wide range of instruments with the appropriate filters. However, a preferred imaging method is confocal microscopy with 20x and 63x (oil) objectives; refer to the discussion for the reasons. Refer to Figure 2 for the determination of the optimal signal-to-noise ratio.
<ol>
	<li>Use a confocal microscope equipped with 488, 561, and 633 nm laser lines. Use the following acquisition parameters (see Table 1): Opal 690 (HeNe 633 nm, detection range 668-696 nm, power 2.0, gain 724); Opal 570 (DPSS laser 561 nm, detection range 579-627 nm, &#60;power 15.0, &#60;gain 450); Opal 520 (argon laser 488 nm, detection range 499-535 nm , &#60;power 6.0, &#60;gain 830); DAPI (diode laser 405, detection range 415-502 nm, &#60;power 12.0, &#60;gain 532).</li>
	<li>To improve the quality of the images, acquire with a line averaging of 4 and a pixel size of at least 1024 x 1024. 
	NOTE: The above parameters are only provided as an example, and modifications are recommended depending on one instrument, magnification (20x or 63x), expression levels, and endogenous levels of fluorescence for any given tissue.</li>
	<li>Once satisfied with the acquisition parameters, collect images using the same settings. Attribute false colors to each channel to facilitate visualization. 
	NOTE: For example, red (Phox2b or Prdm12), cyan (Cck1r), yellow (Ghsr), and grey (DAPI).</li>
	<li>Perform cell counting using the digital images by identifying the outline of RNAscope-positive profiles with one nucleus lightly stained with DAPI. Calculate the percentage of RNAscope-positive profiles expressing each transcript [Figure 3A-C].</li>
	<li>To improve the rigor and accuracy of the estimates, count at least 2,000 neuronal profiles from at least 4 different animals. Perform counting from left and right ganglia separately.</li>
	<li>Input the data in a pie chart with the total number of counted profiles.</li>
	<li>Use imaging software to acquire images and stitch together 20x images. Use ImageJ-Fiji and another photo and design software to generate the final plates. Apply adjustments to contrast and lightness uniformly to all images.</li>
</ol>

The authors declare no competing financial interests.

The technique of ISH was invented in the late 1960s42. However, it is not until the mid-1980s that it was applied for the detection of mRNAs in the central and peripheral nervous systems43,44. Considering the heterogeneity of the nervous system and recurring issues with antibodies, localizing a particular transcript at the cellular level remains an invaluable tool. Nonetheless, traditional ISH methods have remained laborious and variably s...

The cell bodies of vagal afferents are contained in the jugular, petrosal, and nodose ganglia1,2,3. Their axons travel together via several branches of the vagus nerve to craniocervical, thoracic, and abdominal territories4,5,6,7. From their visceral endings, vagal afferents can respond to a wide range of physiological and noxious stimuli8,9,10. However, the distribution of signaling molecules and receptors involved in vagal sensing remains poorly characterized. This is partly because the vagal ganglia, in spite of their small size, express a broad spectrum of receptors, including a large number of GPCRs8,11,12,13. Moreover, vagal afferent neurons are inherently heterogeneous and display distinct molecular profiles14. To complicate the matter, the jugular, petrosal, and nodose ganglia are attached in the mouse, thereby forming a single ganglionic mass. Lastly, in a subset of animals, the nodose ganglion is attached to the sympathetic superior cervical ganglion15.
In the past, investigators have turned to immunohistochemistry to study the neurochemical make-up of vagal afferent neurons16,17,18. While immunohistochemistry using validated antibodies is useful, the results of immunohistochemical studies must be interpreted with caution. For example, numerous efforts to identify specific antibodies against GPCRs have failed19,20,21,22,23,24,25, leading investigators to conclude that the majority of antibodies against GPCRs are unreliable. To circumvent these issues, quantitative PCR (qPCR) has been widely used for assessing gene expression in the rodent vagal ganglionic mass26,27,28,29. However, examining gene expression using qPCR occurs at the cost of a loss of spatial information. In particular, it cannot be predicted how many cells or what cell type(s) express a particular gene of interest (e.g., nodose vs. jugular cells). Recurring issues also include the contamination with adjacent tissues and the inclusion of variable lengths of the vagus nerve, superior cervical, and jugular ganglia during dissection15. As a result of the above difficulties, controversy surrounds the expression and distribution of several GPCRs in vagal afferent neurons. One particularly puzzling example relates to the ghrelin receptor (Ghsr). Whereas some studies have found widespread expression of this receptor in vagal afferent neurons30,31,32, others have found Ghsr mRNA to be nearly undetectable in the nodose ganglion11,14. Detailed mapping of Ghsr mRNA in the vagal ganglionic mass is therefore warranted.
In situ hybridization (ISH) has also been used to assess gene expression patterns in the vagal ganglionic mass7,11,12,33,34,35. Because RNA-based techniques remain more reliable and specific than antibody-based techniques under most circumstances36,37, ISH studies have proven valuable for better understanding of the neurochemical coding of vagal afferent neurons. Nonetheless, traditional ISH techniques themselves are not without caveats. Radioactive ISH is sensitive but generates background and remains cumbersome38. Non-radioactive ISH is less complicated but also less sensitive38. In contrast, the recently developed RNAscope ISH method is highly sensitive and generates minimal background39. The current study applied multiplex fluorescent RNAscope to the detection of GPCRs in vagal afferent neurons of the mouse. We focused on mapping the distribution of Ghsr and compared its distribution to that of the cholecystokinin receptor (Cck1r), another GPCR well known to be expressed in the nodose ganglion34. Lastly, the two transcription factors, paired-like homeobox 2b (Phox2b) and PR domain zinc finger protein 12 (Prdm12), were used as selective markers for nodose and jugular afferent neurons, respectively14. Without visualizing Phox2b or Prdm12, it would be challenging to identify jugular vs. nodose afferents with certainty. Potential technical pitfalls are also discussed throughout the article.

<table><tbody><tr><td>10x PBS</td><td>Fisher Scientific</td><td>BP399-4</td><td></td></tr><tr><td>20x SSC</td><td>Invitrogen</td><td>AM9763</td><td></td></tr><tr><td>-80&amp;deg;C freezer</td><td>PHCBI</td><td>MDF-DU901VHA-PA</td><td></td></tr><tr><td>Adobe Photoshop 2021</td><td>Adobe</td><td></td><td>photo and design software</td></tr><tr><td>Baking oven</td><td>Thermo Scientific</td><td>Model:658</td><td></td></tr><tr><td>Confocal microscope</td><td>Zeiss</td><td>LSM880 Airyscan</td><td></td></tr><tr><td>Cover glass</td><td>Brain Research Laboratories</td><td>2460-1.5D</td><td></td></tr><tr><td>Cryostat</td><td>Leica</td><td>CM 3050 S</td><td></td></tr><tr><td>Dumont #5 Forceps</td><td>F.S.T.</td><td>11252-20</td><td></td></tr><tr><td>Ecomount</td><td>Biocare Medical</td><td>EM 897L</td><td>mounting medium</td></tr><tr><td>HybEZ oven</td><td></td><td></td><td>hybridization oven</td></tr><tr><td>Hydrophobic pen</td><td>Vector Laboratories</td><td>H-4000</td><td></td></tr><tr><td>ImageJ-Fiji</td><td>NIH</td><td></td><td></td></tr><tr><td>Large scissors</td><td>Henry Schein</td><td>100-7561</td><td></td></tr><tr><td>Micro centrifuge tubes</td><td>VWR</td><td>20170-333</td><td></td></tr><tr><td>Minipump variable flow</td><td>Fisher Scientific</td><td>13-876-1</td><td></td></tr><tr><td>Opal 520</td><td>Akoya biosciences</td><td>FP1 1487001KT</td><td>Fluorescent biomarker</td></tr><tr><td>Opal 570</td><td>Akoya biosciences</td><td>FP1 1488001KT</td><td>Fluorescent biomarker</td></tr><tr><td>Opal 690</td><td>Akoya biosciences</td><td>FP1 1497001KT</td><td>Fluorescent biomarker</td></tr><tr><td>ProLong Gold Antifade Mountant</td><td></td><td></td><td>mounting medium for fluorescently labeled cells</td></tr><tr><td>RNAscope Multiplex Fluorescent Reagent Kit v2</td><td>ACD /Bio-Techne</td><td>323100</td><td>multiplex kit</td></tr><tr><td>RNAscope probe Mouse Cck1r-C3</td><td>ACD /Bio-Techne</td><td>313751-C3</td><td></td></tr><tr><td>RNAscope probe Mouse DapB</td><td>ACD /Bio-Techne</td><td>310043</td><td></td></tr><tr><td>RNAscope probe Mouse Ghsr</td><td>ACD /Bio-Techne</td><td>426141</td><td></td></tr><tr><td>RNAscope probe Mouse Phox2b-C2</td><td>ACD /Bio-Techne</td><td>407861-C2</td><td></td></tr><tr><td>RNAscope probe Mouse Prdm12-C2</td><td>ACD /Bio-Techne</td><td>524371-C2</td><td></td></tr><tr><td>RnaseZap</td><td>Sigma</td><td>R2020</td><td>Rnase decontaminating solution</td></tr><tr><td>Small dissecting scissors</td><td>Millipore Sigma</td><td>Z265977</td><td></td></tr><tr><td>Superfrost Plus slides</td><td>Fisherbrand</td><td>1255015</td><td></td></tr><tr><td>Tissue Tek OCT medium</td><td>Sakura</td><td>4583</td><td></td></tr><tr><td>User manual</td><td>ACD</td><td>323100 USM</td><td></td></tr><tr><td>Vannas Spring Scissors</td><td>Roboz</td><td>RS 5620</td><td></td></tr><tr><td>ZEN Imaging Software</td><td>Zeiss</td><td></td><td></td></tr></tbody></table>

detection of g protein-coupled receptor expression in mouse vagal afferent neurons using multiplex in situ hybridization

This study describes a protocol for the multiplex in situ hybridization (ISH) of the mouse jugular-nodose ganglia, with a particular emphasis on detecting the expression of G protein-coupled receptors (GPCRs). Formalin-fixed jugular-nodose ganglia were processed with the RNAscope technology to simultaneously detect the expression of two representative GPCRs (cholecystokinin and ghrelin receptors) in combination with one marker gene of either nodose (paired-like homeobox 2b, Phox2b) or jugular afferent neurons (PR domain zinc finger protein 12, Prdm12). Labeled ganglia were imaged using confocal microscopy to determine the distribution and expression patterns of the aforementioned transcripts. Briefly, Phox2b afferent neurons were found to abundantly express the cholecystokinin receptor (Cck1r) but not the ghrelin receptor (Ghsr). A small subset of Prdm12 afferent neurons was also found to express Ghsr and/or Cck1r. Potential technical caveats in the design, processing, and interpretation of multiplex ISH are discussed. The approach described in this article may help scientists in generating accurate maps of the transcriptional profiles of vagal afferent neurons.

While RNAScope can be applied to animals of any age, sex, or genetic background, it is advisable to work with young adults (&#60;3 months old). This is because fluorescent artifacts (e.g., lipofuscin) are common findings in neurons of older animals41. The formalin-fixed ganglia from older mice often contain surprisingly intense endogenous fluorescence that can easily be mistaken for genuine staining (Figure 1A,B). In any case, it is advisable to verif...

Watch this Scientific Journal Video about Detection of G Protein-coupled Receptor Expression in Mouse Vagal Afferent Neurons using Multiplex In Situ Hybridization at JoVE.com

Detection of G Protein-coupled Receptor Expression in Mouse Vagal Afferent Neurons using Multiplex In Situ Hybridization

Multiplex in situ hybridization (ISH) was employed to simultaneously visualize the transcripts for two G protein-coupled receptors and one transcription factor in the entire vagal ganglionic complex of the adult mouse. This protocol could be used to generate accurate maps of the transcriptional profiles of vagal afferent neurons.

Detection of G Protein-coupled Receptor Expression in Mouse Vagal Afferent Neurons using Multiplex In Situ Hybridization

This study describes a protocol for the multiplex in situ hybridization (ISH) of the mouse jugular-nodose ganglia, with a particular emphasis on detecting ...

detection-g-protein-coupled-receptor-expression-mouse-vagal-afferent

Research

JoVE Journal

Neuroscience

3.8K Views.  UTSouthwestern Medical Center at Dallas. Deze procedure is zeer gevoelig en maakt een visualisatie van verschillende transcripties met minimale achtergrond mogelijk. Deze methode kan nauwkeurige kaarten genereren van het transcriptionele profiel van zeer heterogene celtypen. De procedure wordt gedemonstreerd door mij en Johnson Bob-manuel, een onderzoekstechnicus in het laboratorium.Begin met het verzamelen van de volledige vagale ganglionische massa van de acht weken oude muis van elke kant, met behulp van een ontleedbare sc...

Video: Detection of G Protein-coupled Receptor Expression in Mouse Vagal Afferent Neurons using Multiplex In Situ Hybridization

Tissue-specific miRNA Expression Profiling in Mouse Heart Sections Using In Situ Hybridization

micro-RNAs (miRNAs) are single-stranded RNA transcripts that bind to messenger RNAs (mRNAs) and inhibit their translation or promote their degradation. To date, miRNAs have been implicated in a large number of biological and disease processes, which has signified the need for the reliable detection methods of miRNA transcripts. Here, we describe a detailed protocol for digoxigenin-labeled (DIG) Locked Nucleic Acid (LNA) probe-based miRNA detection, combined with protein immunostaining on mouse heart sections. First, we performed an in situ hybridization technique using the probe to identify miRNA-182 expression in heart sections from control and cardiac hypertrophy mice. Next, we performed immunostaining for cardiac Troponin T (cTnT) protein, on the same sections, to co-localize miRNA-182 with the cardiomyocyte cells. Using this protocol, we were able to detect miRNA-182 through an alkaline phosphatase based colorimetric assay, and cTnT through fluorescent staining. This protocol can be used to detect the expression of any miRNA of interest through DIG-labeled LNA probes, and relevant protein expression on mouse heart tissue sections.

Tissue-specific miRNA Expression Profiling in Mouse Heart Sections Using In Situ Hybridization

micro-RNAs (miRNAs) are short and highly homologous RNA sequences, serving as post-transcriptional regulators of messenger RNAs (mRNAs). Current miRNA detection methods vary in sensitivity and specificity. We describe a protocol that combines in situ hybridization and immunostaining for concurrent detection of miRNA and protein molecules on mouse heart tissue sections.

micro-RNAs (miRNAs) are single-stranded RNA transcripts that bind to messenger RNAs (mRNAs) and inhibit their translation or promote their degradation. To ...

Genetics

Imaging Pheromone Sensing in a Mouse Vomeronasal Acute Tissue Slice Preparation

Peter Karlson and Martin L&uuml;scher used the term pheromone for the first time in 19591 to describe chemicals used for intra-species communication. Pheromones are volatile or non-volatile short-lived molecules2 secreted and/or contained in biological fluids3,4, such as urine, a liquid known to be a main source of pheromones3. Pheromonal communication is implicated in a variety of key animal modalities such as kin interactions5,6, hierarchical organisations3 and sexual interactions7,8 and are consequently directly correlated with the survival of a given species9,10,11. In mice, the ability to detect pheromones is principally mediated by the vomeronasal organ (VNO)10,12, a paired structure located at the base of the nasal cavity, and enclosed in a cartilaginous capsule. Each VNO has a tubular shape with a lumen13,14 allowing the contact with the external chemical world. The sensory neuroepithelium is principally composed of vomeronasal bipolar sensory neurons (VSNs)15. Each VSN extends a single dendrite to the lumen ending in a large dendritic knob bearing up to 100 microvilli implicated in chemical detection16. Numerous subpopulations of VSNs are present. They are differentiated by the chemoreceptor they express and thus possibly by the ligand(s) they recognize17,18. Two main vomeronasal receptor families, V1Rs and V2Rs19,20,21,22, are composed respectively by 24023 and 12024 members and are expressed in separate layers of the neuroepithelium. Olfactory receptors (ORs)25 and formyl peptide receptors (FPRs)26,27 are also expressed in VSNs.
Whether or not these neuronal subpopulations use the same downstream signalling pathway for sensing pheromones is unknown. Despite a major role played by a calcium-permeable channel (TRPC2) present in the microvilli of mature neurons28 TRPC2 independent transduction channels have been suggested6,29. Due to the high number of neuronal subpopulations and the peculiar morphology of the organ, pharmacological and physiological investigations of the signalling elements present in the VNO are complex.
Here, we present an acute tissue slice preparation of the mouse VNO for performing calcium imaging investigations. This physiological approach allows observations, in the natural environment of a living tissue, of general or individual subpopulations of VSNs previously loaded with Fura-2AM, a calcium dye. This method is also convenient for studying any GFP-tagged pheromone receptor and is adaptable for the use of other fluorescent calcium probes. As an example, we use here a VG mouse line30, in which the translation of the pheromone V1rb2 receptor is linked to the expression of GFP by a polycistronic strategy.

In mice, the ability to detect pheromones is principally mediated by the vomeronasal organ (VNO). Here, an acute tissue slice preparation of VNO for performing calcium imaging is described. This physiological approach allows observations of subpopulations and/or individual neurons in a living tissue and is convenient for receptor-ligand identification.

Peter Karlson and Martin L&uuml;scher used the term pheromone for the first time in 19591 to describe chemicals used for intra-species communication. ...

A Method to Target and Isolate Airway-innervating Sensory Neurons in Mice

Somatosensory nerves transduce thermal, mechanical, chemical, and noxious stimuli caused by both endogenous and environmental agents. The cell bodies of these afferent neurons are located within the sensory ganglia. Sensory ganglia innervate a specific organ or portion of the body. For instance, the dorsal root ganglia (DRG) are located in the vertebral column and extend processes throughout the body and limbs. The trigeminal ganglia are located in the skull and innervate the face, and upper airways. Vagal afferents of the nodose ganglia extend throughout the gut, heart, and lungs. The nodose neurons control a diverse array of functions such as: respiratory rate, airway irritation, and cough reflexes. Thus, to understand and manipulate their function, it is critical to identify and isolate airway specific neuronal sub-populations. In the mouse, the airways are exposed to a fluorescent tracer dye, Fast Blue, for retrograde tracing of airway-specific nodose neurons. The nodose ganglia are dissociated and fluorescence activated cell (FAC) sorting is used to collect dye positive cells. Next, high quality ribonucleic acid (RNA) is extracted from dye positive cells for next generation sequencing. Using this method airway specific neuronal gene expression is determined.

Organ specific sensory neurons are difficult to identify. Fast Blue tracing is used to identify nodose neurons innervating the airways for cell sorting. Sorted nodose neurons are used to extract high quality ribonucleic acid (RNA) for sequencing. Using this protocol, gene expression of airway specific neurons is determined.

Somatosensory nerves transduce thermal, mechanical, chemical, and noxious stimuli caused by both endogenous and environmental agents. The cell bodies of ...

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 ...

In-depth Physiological Analysis of Defined Cell Populations in Acute Tissue Slices of the Mouse Vomeronasal Organ

In most mammals, the vomeronasal organ (VNO) is a chemosensory structure that detects both hetero- and conspecific social cues. Vomeronasal sensory neurons (VSNs) express a specific type of G protein-coupled receptor (GPCR) from at least three different chemoreceptor gene families allowing sensitive and specific detection of chemosensory cues. These families comprise the V1r and V2r gene families as well as the formyl peptide receptor (FPR)-related sequence (Fpr-rs) family of putative chemoreceptor genes. In order to understand the physiology of vomeronasal receptor-ligand interactions and downstream signaling, it is essential to identify the biophysical properties inherent to each specific class of VSNs.
The physiological approach described here allows identification and in-depth analysis of a defined population of sensory neurons using a transgenic mouse line (Fpr-rs3-i-Venus). The use of this protocol, however, is not restricted to this specific line and thus can easily be extended to other genetically modified lines or wild type animals.

Here, we describe a physiological approach that allows identification and in-depth analysis of a defined population of sensory neurons in acute coronal tissue slices of the mouse vomeronasal organ using whole-cell patch-clamp recordings.

In most mammals, the vomeronasal organ (VNO) is a chemosensory structure that detects both hetero- and conspecific social cues. Vomeronasal sensory ...

Single Cell Multiplex Reverse Transcription Polymerase Chain Reaction After Patch-clamp

The cerebral cortex is composed of numerous cell types exhibiting various morphological, physiological, and molecular features. This diversity hampers easy identification and characterization of these cell types, prerequisites to study their specific functions. This article describes the multiplex single cell reverse transcription polymerase chain reaction (RT-PCR) protocol, which allows, after patch-clamp recording in slices, to detect simultaneously the expression of tens of genes in a single cell. This simple method can be implemented with morphological characterization and is widely applicable to determine the phenotypic traits of various cell types and their particular cellular environment, such as in the vicinity of blood vessels. The principle of this protocol is to record a cell with the patch-clamp technique, to harvest and reverse transcribe its cytoplasmic content, and to detect qualitatively the expression of a predefined set of genes by multiplex PCR. It requires a careful design of PCR primers and intracellular patch-clamp solution compatible with RT-PCR. To ensure a selective and reliable transcript detection, this technique also requires appropriate controls from cytoplasm harvesting to amplification steps. Although precautions discussed here must be strictly followed, virtually any electrophysiological laboratory can use the multiplex single cell RT-PCR technique.

This protocol describes the critical steps and precautions required to perform single cell multiplex reverse transcription polymerase chain reaction after patch-clamp. This technique is a simple and effective method to analyze the expression profile of a predetermined set of genes from a single cell characterized by patch-clamp recordings.

The cerebral cortex is composed of numerous cell types exhibiting various morphological, physiological, and molecular features. This diversity hampers ...

Combining Multiplex Fluorescence In Situ Hybridization with Fluorescent Immunohistochemistry on Fresh Frozen or Fixed Mouse Brain Sections

Fluorescent in situ hybridization (FISH) is a molecular technique that identifies the presence and spatial distribution of specific RNA transcripts within cells. Neurochemical phenotyping of functionally identified neurons usually requires concurrent labelling with multiple antibodies (targeting protein) using immunohistochemistry (IHC) and optimization of in situ hybridization (targeting RNA), in tandem. A &#34;neurochemical signature&#34; to characterize particular neurons may be achieved however complicating factors include the need to verify FISH and IHC targets before combining the methods, and the limited number of RNAs and proteins that may be targeted simultaneously within the same tissue section.
Here we describe a protocol, using both fresh frozen and fixed mouse brain preparations, which detects multiple mRNAs and proteins in the same brain section using RNAscope FISH followed by fluorescence immunostaining, respectively. We use the combined method to describe the expression pattern of low abundance mRNAs (e.g., galanin receptor 1) and high abundance mRNAs (e.g., glycine transporter 2), in immunohistochemically identified brainstem nuclei.
Key considerations for protein labelling downstream of the FISH assay extend beyond tissue preparation and optimization of FISH probe labelling. For example, we found that antibody binding and labelling specificity can be detrimentally affected by the protease step within the FISH probe assay. Proteases catalyze hydrolytic cleavage of peptide bonds, facilitating FISH probe entry into cells, however they may also digest the protein targeted by the subsequent IHC assay, producing off target binding. The subcellular location of the targeted protein is another factor contributing to IHC success following FISH probe assay. We observed IHC specificity to be retained when the targeted protein is membrane bound, whereas IHC targeting cytoplasmic protein required extensive troubleshooting. Finally, we found handling of slide-mounted fixed frozen tissue more challenging than fresh frozen tissue, however IHC quality was overall better with fixed frozen tissue, when combined with RNAscope.

Combining Multiplex Fluorescence In Situ Hybridization with Fluorescent Immunohistochemistry on Fresh Frozen or Fixed Mouse Brain Sections

This protocol describes a method for combining fluorescence in situ hybridization (FISH) and fluorescence immunohistochemistry (IHC) in both fresh frozen and fixed mouse brain sections, with the goal of achieving multilabel FISH and fluorescence IHC signal. IHC targeted cytoplasmic and membrane attached proteins.

Fluorescent in situ hybridization (FISH) is a molecular technique that identifies the presence and spatial distribution of specific RNA transcripts within ...

Investigation of Activated Mouse Olfactory Sensory Neurons via Combined Immunostaining and in situ Hybridization

Animals rely on chemical communication to convey and perceive relevant environmental information, ranging from assessment of food quality to detection of available mating partners or threats. In mice, this task is executed primarily by the olfactory system and its underlying subsystems, including the main and accessory olfactory systems. Both have peripheral organs populated by sensory neurons expressing G-protein coupled receptors able to bind chemical cues that reach the nasal cavity. Even though the molecular characteristics of these receptors is well understood, little is known about their cognate specific ligands. The method described here combines in situ hybridization detection of olfactory or vomeronasal receptors with immunodetection of phosphorylated ribosomal protein S6 (pS6) - a marker of neuronal activation. This protocol was devised to identify neurons activated after a single event of exposure to purified or complex chemical stimuli detected by the olfactory organs. Importantly, this technique allows the investigation of neurons triggered in biologically relevant contexts. Ideally, this method should be used to probe the molecular biology of the olfactory system and to study olfactory behaviors.

Investigation of Activated Mouse Olfactory Sensory Neurons via Combined Immunostaining and in situ Hybridization

This protocol combines in situ hybridization with immunofluorescence to identify olfactory and vomeronasal receptor genes expressed in olfactory sensory neurons after activation by chemical stimuli in the mouse.

Animals rely on chemical communication to convey and perceive relevant environmental information, ranging from assessment of food quality to detection of ...

Multiplex Detection of Gene Expression in the Intact Drosophila Brain Using Expansion-Assisted Iterative Fluorescence In Situ Hybridization

Understanding gene expression is essential for deciphering cellular functions. However, methods for analyzing the expression of numerous genes in situ within a given tissue remain limited. The EASI-FISH protocol described here has been adapted to detect the expression of dozens of genes in the intact adult Drosophila central nervous system (CNS) using commercially available reagents. This protocol includes a new gel formulation that enhances gel robustness, enabling multiple rounds of hybridization and allowing the embedding of multiple brains per gel. This improvement increases throughput, facilitates optimal comparison of experimental conditions, and reduces reagent costs. Additionally, by employing the GAL4-UAS system for co-detection of green fluorescent protein (GFP), gene expression can be visualized in specific neuronal or glial cell types. Notably, the high resolution achieved through expansion microscopy, combined with the sensitivity of the method, allows for the detection of single RNA transcripts. This approach effectively integrates high image quality with high throughput, making it a powerful tool for studying gene expression throughout the intact fly brain.

Expansion-Assisted Iterative Fluorescence In Situ Hybridization (EASI-FISH) is a robust technique that combines expansion microscopy with fluorescence in situ hybridization (FISH) to detect the expression of multiple genes in thick tissue. This protocol outlines recent advancements in the method and its adaptation for labeling the intact Drosophila central nervous system..

Understanding gene expression is essential for deciphering cellular functions. However, methods for analyzing the expression of numerous genes in situ ...

 Performing Double Fluorescence In Situ Hybridization to Detect Gene Expression in Mouse Brain Sections

<a href='https://app.jove.com/v/53976/combining-double-fluorescence-situ-hybridization-with-immunolabelling'>Source: Jolly, S., et.al. Combining Double Fluorescence In Situ Hybridization with Immunolabelling for Detection of the Expression of Three Genes in Mouse Brain Sections. J. Vis. Exp. (2016).</a>This video demonstrates the use of double fluorescence in situ hybridization to visualize the expression of two RNA targets in mouse brain sections. The protocol involves sequential detection of RNA using probes labeled with fluorescein isothiocyanate and digoxigenin, followed by enzymatic signal amplification to enhance sensitivity. The resulting fluorescence enables co-localization analysis of the target RNAs within the tissue.

Source: Jolly, S., et.al. Combining Double Fluorescence In Situ Hybridization with Immunolabelling for Detection of the Expression of Three Genes in Mouse ...

Detection of G Protein-coupled Receptor Expression in Mouse Vagal Afferent Neurons using Multiplex In Situ Hybridization

Samenvatting

Meer video's verkennen