This work was funded by Australian Research Council Discovery Project grant DP180101890 and Rebecca L Cooper Medical Research Foundation project grant PG2018110

A summary of tissue pre-processing steps may be found in Figure 1. All procedures were carried out in compliance with the Animal Care and Ethics Committee of the University of New South Wales in accordance with the guidelines for the use and care of animals for scientific purposes (Australian National Health and Medical Research Council).
1. Sample preparation of fresh frozen brain tissue
<ol>
	<li>Transcardial Perfusion
	<ol>
		<li>Prepare heparinized (2500 U/L) 0.1 M phosphate buffer (PB), pH 7.5. Make dry ice ethanol slurry by mixing dry ice with ethanol. This will have a temperature of approximately −72 °C and will be used for immediate freezing of the harvested tissue.</li>
		<li>Euthanize adult C57BL/6 and Phox2b-GFP11 (Mouse Genome Informatics database ID MGI:5776545) mice by anesthetizing with sodium pentobarbital (70 mg/kg, i.p.), using a 27.5 inch needle gauge. 
		CAUTION: Pentobarbital is a barbiturate. It is acutely toxic in high doses and may cause death by respiratory arrest. Consult local healthcare, legal and material safety guidelines before use.</li>
		<li>Expose the heart and cannulate the left ventricle with a drawing-up needle (23 inch gauge). Perform transcardial perfusion with heparinized 0.1 M PB until the blood clears (2-3 minutes) at a flow rate of 11-13 mL/min. Determine blood clearing by monitoring the coloration of the liver and the effusate from the right atrium12.</li>
		<li>Isolate the brain from the skull cavity, immediately embed it in Optimal Cutting Temperature Compound (OCT) in a cryomold or aluminum foil and place it on the dry ice ethanol bath. Store the frozen embedded tissue in an airtight container at - 80 °C for up to 3 months.</li>
	</ol>
	</li>
	<li>Sectioning of fresh frozen tissue
	<ol>
		<li>Set the cryostat temperature to -20 °C. Leave the OCT-embedded tissue and a cryostat chuck in the cryostat for ~30 minutes to allow for equilibration to the new temperature. 
		NOTE: Keep the tissue frozen at all times; transport the tissue from the -80 °C freezer to the cryostat on dry ice.</li>
		<li>Secure the tissue to the pre-chilled cryostat chuck using OCT compound. In this protocol, tissue blocks were mounted onto the chuck in the coronal plane. 
		NOTE: Trim excess OCT from the tissue, using a razor blade, to minimize the amount of OCT being cut by the cryostat and subsequently transferred onto the glass slide.</li>
		<li>Cut 14 µm thick coronal sections and mount them onto charged glass microscopy slides.
		<ol>
			<li>Warm the slides to room temperature before mounting the sections. Once the section has been mounted, keep the slides in a slide box in the cryostat.</li>
			<li>If more than one section needs to be mounted on one slide, warm the area for the second section by placing a finger on the opposite side of the slide for 5-10 seconds to aid adherence of the section to the slide. A cold tissue section will not attach to a cold slide. The sections should adhere to the slides flat; folding will cause them to fall off the slides during wash steps.</li>
			<li>If cracks are noticed in the sections, increase the cryostat temperature by 1-5 °C to avoid this. It is particularly important to place tissue sections in close proximity to one another on the same slide. This will prevent wastage of FISH probes and reagents during the assay.</li>
		</ol>
		</li>
		<li>Store tissue sections mounted onto glass slides in an air-tight container at -80 °C for up to 6 months. 
		NOTE: Keep the sections frozen at all times and avoid freeze thaw cycles, to prevent RNA degradation. Transport the slide box from inside the cryostat to the -80 °C freezer on dry ice.</li>
	</ol>
	</li>
	<li>Fixation of fresh frozen tissue
	<ol>
		<li>On the day the FISH probe assay is to be performed, prepare 4% paraformaldehyde (PFA) in 0.1 M PB, pH 7.5 (4% PFA solution). Filter by passing through filter paper (Grade 1: 11 µm, Table of Materials) in a Buchner funnel or crucible filter. 
		CAUTION : PFA is harmful and toxic by skin contact or inhalation. All procedures with PFA solution should be performed in a fume hood cabinet. PFA solution waste should be disposed of carefully following institutional safety protocols.</li>
		<li>Cool the 4% PFA solution to 4 °C. Transport the slide-mounted tissue from the -80 °C freezer in dry ice and immediately immerse it in the pre-chilled fixative for 15 minutes. 
		NOTE: It is important that this fixation step does not exceed 15 minutes as over-fixation will result in non-specific background labelling.</li>
	</ol>
	</li>
	<li>Dehydration of fresh frozen tissue
	<ol>
		<li>Dehydrate tissue sections by submerging the slides in graded concentrations of ethanol. In a Coplin jar, first submerge in 50%, then 70% and finally absolute ethanol, for 5 minutes each at room temperature. Repeat the final absolute ethanol incubation a second time.</li>
		<li>Air dry slides and, outline the group of sections using a hydrophobic barrier pen, ensuring that the internal area is kept to a minimum. 
		NOTE: Make sure that the glass slide is completely dry before drawing the hydrophobic barrier. The hydrophobic barrier should surround the tissue sections completely without gaps and must be dry before further processing.</li>
	</ol>
	</li>
</ol>
2. Sample preparation of fixed frozen brain tissue
<ol>
	<li>Transcardial perfusion fixation
	<ol>
		<li>Euthanize mice by anesthetizing with sodium pentobarbital (70 mg/kg, i.p) followed by transcardial perfusion, first with 0.1 M PB then 4% PFA solution. Fix with 10 minutes of perfusion at 11-13 mL/min.</li>
		<li>Isolate the brain from the skull cavity following perfusion-fixation and submerge overnight in 4% PFA solution, at 4 °C.</li>
	</ol>
	</li>
	<li>Tissue sectioning of fixed tissue
	<ol>
		<li>Rinse the brain in sterile 0.1 M phosphate buffered saline (PBS) before removing the meningeal layers, with the aid of a dissecting microscope, using fine forceps.</li>
		<li>Cut the brain precisely into blocks (separate the brainstem from the forebrain prior to vibratome sectioning) using a brain matrix (Table of Materials). Specifically, cut the brainstem caudally at the pyramidal decussation and dissect away the cerebellum. Similarly, cut the forebrain immediately rostral to the optic chiasm.</li>
		<li>Secure the tissue onto a vibrating microtome chuck using cyanoacrylate and embed in 2% agar solution.</li>
		<li>Cut 30 µm thick tissue sections using a vibrating microtome and store cut sections in cryoprotectant solution (30% RNase free sucrose, 30% ethylene glycol, 1% polyvinylpyrrolidone (PVP-40), in 0.1 M PB, pH 7.4). Tissue sections may be stored in cryoprotectant at -20 °C for up to 6 months.</li>
	</ol>
	</li>
	<li>Preparation of fixed sections prior to FISH
	<ol>
		<li>On the day of FISH, wash free floating sections three times, for 10 minutes per wash, to remove the cryoprotectant solution. To wash, place sections in 0.1 M PBS in a 12 well cell culture plate and agitate on a rotating platform shaker (90 - 100 rpm).</li>
		<li>After washes, use a paintbrush to mount sections on glass microscopy slides and air dry for at least 2 hours. 
		NOTE: The sections should adhere flat onto the slides as any pronounced folds will cause them to detach during washes.</li>
		<li>Using a hydrophobic barrier pen, draw a barrier around the sections to restrict the FISH reagents to the sections. Once more, it is important to minimize the internal area of the outline drawn with the barrier pen. 
		​POSSIBLE BREAK POINT: The sections could be stored at room temperature, overnight, to continue the assay the next day.</li>
	</ol>
	</li>
</ol>
3. FISH assay
NOTE: The rest of the protocol applies to both fresh frozen and fixed frozen tissue.
<ol>
	<li>Prepare the reagents and instruments for hybridization and amplification steps.
	<ol>
		<li>Set a benchtop incubator and water bath to 40 °C.</li>
		<li>Prepare a humidified, light-protected chamber for incubating slides. Humidification prevents drying of tissues - slides are securely located above a moist reservoir. Ideally, the chamber is made of heavy-duty polystyrene, it is light-proof and airtight to maintain a saturated water vapor atmosphere. Closure of the chamber relies on minimal friction to avoid movement. We used a slide box lined with wet laboratory wipes (Table of Materials) at the bottom. Place the slide box inside the incubator to prewarm it to 40 °C.</li>
		<li>Warm the 50x Wash Buffer (Table of Materials) and probes to 40 °C for 10 minutes, using the water bath, then cool to room temperature.</li>
		<li>Prepare 1 L of 1x Wash Buffer from the 50x stock concentration.</li>
		<li>Prepare probe mixture (Table of Materials): the C1 probe is ready to use at stock concentration whereas C2 and C3 probes are shipped as 50x concentration and require dilution with the diluent supplied in the kit. 
		NOTE: Probe mixtures can be stored at 4 °C for up to 6 months.</li>
	</ol>
	</li>
	<li>Protease treatment
	<ol>
		<li>Incubate sections with Protease III (Table of Materials) at room temperature for 30 minutes. 
		NOTE: Ensure that Protease III and incubation reagents in downstream processes (probe mixture, amplification solutions, blocking buffer and antibody sera) cover the sections entirely. A pipet tip may be used to spread the reagent onto the section to cover the entire area inside the hydrophobic barrier.</li>
		<li>Wash slides twice with 0.1 M PBS, for 2 min each time, in a large plastic square Petri dish. Here a 245 mm x 245 mm square bioassay dish was used (Table of Materials). Hold from one side of the dish and tilt gently 3-5 times. After washes, flick excess 0.1 M PBS from slide and immediately add the next reagent. Do not let tissue sections dry. 
		NOTE: During each wash, the slides are immersed in solution at room temperature. This is the workflow for all subsequent wash steps. The fixed 30 µm thick sections dislodge from slides more easily than 14 µm thick sections, be gentle during the washes.</li>
	</ol>
	</li>
	<li>Hybridization and amplification
	<ol>
		<li>After washing off the protease solution, place the slides in the humidified, prewarmed chamber. Incubate sections with probe mixture (Table of Materials) for 2 hours at 40 °C inside a benchtop incubator. 
		​NOTE: Ensure there are at least 2 sections set aside for positive and negative control probes to assess sample RNA quality and optimal permeabilization. Positive control probes target house-keeping genes; here, these were a cocktail of RNAs targeting ubiquitin C (UBC; high-abundance), peptidylpropyl isomerase B (PPIB; moderate-abundance) and RNA polymerase 2a (POLR2A; low-abundance). Negative control probes target the bacterial 4-hydroxy-tetrahydrodipicolinate reductase (DapB) gene, which is normally absent in mouse brain samples. Positive DapB signal indicates non-specific signal and/or bacterial contamination of the sample.</li>
		<li>Following hybridization with the probe mixture, the signal amplification steps consist of incubation with Amp 1-FL (30 minutes), then with Amp 2-FL (15 minutes), followed by Amp 3-FL (30 minutes) and finally Amp 4-FL (15 minutes) - each at 40 °C. Using the dropper bottles provided, cover tissue sections with amplification solution. Proceed to IHC assay following the last amplification step.</li>
		<li>Rinse slides with Wash Buffer twice for 2 minutes between probe hybridization and each amplification step.</li>
	</ol>
	</li>
</ol>
4. IHC Assay
<ol>
	<li>IHC blocking step
	<ol>
		<li>To prevent non-specific binding of antibodies, incubate the sections for 1 h at room temperature with blocking solution containing 10% normal horse serum, 0.3% Tween20 in 1x TBSm (50 mM Tris-Cl, pH 7.5, 150 mM NaCl, 0.05% merthiolate) following the FISH assay. Prepare primary antibodies in a dilution buffer containing 1x TBSm, 5% normal horse serum and 0.1% Tween20. Primary antibody suppliers are listed in the Table of Materials.</li>
	</ol>
	</li>
	<li>Immunohistochemistry
	<ol>
		<li>Remove excess blocking buffer by flicking the slide and incubate sections with primary antibodies overnight at 4 °C.</li>
		<li>Wash slides 3 times (5 minutes each) with 1x TBSm and incubate with secondary antibody in diluent containing 1x TBSm, 1% normal horse serum and 0.1% Tween20 for 2 hours at room temperature. Secondary antibodies used in this protocol are listed in the Table of Materials.</li>
		<li>Wash slides 3 times with 1x TBSm (5 minutes each) before coverslipping with mounting medium with or without DAPI (Table of Materials).</li>
	</ol>
	</li>
</ol>
5. Imaging
<ol>
	<li>Examine immunostaining under an epifluorescence microscope equipped with a camera (see Table of Material for details). Acquire representative images at 20x magnification and save as TIFF files.</li>
	<li>Export representative images into an image processing software (Table of Materials) for brightness/contrast adjustment to increase the clarity and to reflect true rendering.</li>
</ol>
6. OPTIONAL: Quantitative analysis of target transcripts
NOTE: This is a methods article and quantitative results are not provided. The method of quantification presented here is sourced from Dereli et al.10.
<ol>
	<li>Acquire images from the regions of interest as explained in 5.1 and apply the same microscope and camera settings (such as exposure time and light intensity) to all images of the same fluorophore.</li>
	<li>Plot the neuronal profiles using an image analysis software (Table of Materials).</li>
	<li>Align the sections with reference to Bregma level according to a stereotaxic brain atlas13.</li>
	<li>Apply the same brightness and contrast to all the images of the same fluorophore. Only consider the neurons with DAPI-stained nuclei.</li>
	<li>Manually count the number of mRNA, protein expressing, mRNA/mRNA, protein/protein and mRNA/protein coexpressing cells within the region of interest.</li>
	<li>To decrease bias in the experimental results, have the person quantifying experimental outcomes blinded to the experimental groups.</li>
	<li>Apply Abercrombie correction14 to total cell counts by using the following Abercombie equation: 
	Corrected cell count = manual cell count x section thickness / (section thickness + nuclear size) 
	For example, for 14 µm thick sections, the average nuclear width is calculated to be 7.7 ± 0.3 µm and average section thickness is 14 ± 1 µm based on 30 cells and 10 sections respectively in 5 animals10. According to the Abercrombie equation, corrected cell count would be manual cell count x 14/(14+7.7).</li>
</ol>
<img alt="Tissue processing workflow diagram; shows perfusion, sectioning, fixing, dehydration for FISH." class="xfigimg" src="/files/ftp_upload/61709/61709fig01.jpg" data-altupdatedat="1747911167000" data-alt="Figure 1"> 
Figure 1: Parallel workflow of tissue pre-processing steps for both fresh-frozen and paraformaldehyde fixed tissue. Processing steps for fresh-frozen tissue are displayed in the red outlined boxes, whereas those for paraformaldehyde (PFA) fixed tissue are displayed in the blue outlined boxes. <a href="https://www.jove.com/files/ftp_upload/61709/61709fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="FISH and immunohistochemistry process diagram; probe hybridization, amplification, antibody labeling." class="xfigimg" src="/files/ftp_upload/61709/61709fig02.jpg" data-altupdatedat="1747911167000" data-alt="Figure 2"> 
Figure 2: Summary of combined FISH probe and immunohistochemistry procedure. Following tissue pre-processing, the slide mounted tissue is encircled using a hydrophobic barrier pen, as seen in the first frame, and incubated in a protease solution at room temperature. Following washes, tissue is transferred to a benchtop incubator for hybridization for 2 hours before sequential amplification steps. The in situ hybridization system utilizes a proprietary 'Z probe' design, preamplifiers and amplifiers as seen in frames 3-66. Once tissue has undergone FISH probe processing, it is washed before blocking with normal horse serum. The primary antibody incubation is carried out overnight at 4 °C to maximize antibody-antigen binding. The secondary antibody incubation (2 hours) was carried out at room temperature. <a href="https://www.jove.com/files/ftp_upload/61709/61709fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antigen+retrieval+in+formalin-fixed,+paraffin-embedded+tissues:+an+enhancement+method+for+immunohistochemical+staining+based+on+microwave+oven+heating+of+tissue+sections.">Antigen retrieval in formalin-fixed, paraffin-embedded tissues: an enhancement method for immunohistochemical staining based on microwave oven heating of tissue sections.</a> Journal of Histochemistry and Cytochemistry. 39 (6), 741-748 (1991).</li><li>Yamashita, S., Katsumata, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heat-induced+antigen+retrieval+in+immunohistochemistry:+mechanisms+and+applications.">Heat-induced antigen retrieval in immunohistochemistry: mechanisms and applications.</a> Methods in Molecular Biology. 1560, 147-161 (2017).</li><li>Yamashita, S., Okada, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+heat-induced+antigen+retrieval:+analyses+in+vitro+employing+SDS-PAGE+and+immunohistochemistry.">Mechanisms of heat-induced antigen retrieval: analyses in vitro employing SDS-PAGE and immunohistochemistry.</a> Journal of Histochemistry and Cytochemistry. 53 (1), 13-21 (2005).</li><li>Yamashita, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heat-induced+antigen+retrieval:+mechanisms+and+application+to+histochemistry.">Heat-induced antigen retrieval: mechanisms and application to histochemistry.</a> Progress in Histochemistry and Cytochemistry. 41 (3), 141-200 (2007).</li></ol>

In the neurosciences, FISH and IHC are routinely used to investigate the spatial organization and functional significance of mRNA or proteins within neuronal subpopulations. The protocol described in this study enhances the capacity for simultaneous detection of mRNAs and proteins in brain sections. Our combined multiplex FISH-IHC assay enabled phenotypic identification of distinct neuronal subpopulations in the NTS in both fresh frozen and fixed brain preparations. FISH-IHC in fixed frozen tissue preparations produced r...

Proteins and mRNAs that neurochemically define subpopulations of neurons are commonly identified with a combination of immunohistochemistry (IHC) and/or in situ hybridization (ISH), respectively. Combining ISH with IHC techniques facilitates the characterization of colocalization patterns unique to functional neurons (neurochemical coding) by maximizing multiplex labelling capacity.
Fluorescent ISH (FISH) methods, including RNAscope, have higher sensitivity and specificity compared to earlier RNA detection methods such as radioactive ISH and non-radioactive chromogenic ISH. FISH enables visualization of single mRNA transcripts as punctate stained spots1. Furthermore, the RNAscope assay allows an increased number of RNA targets to be labeled at a time, using different fluorophore tags. Despite these advantages, technical limitations may affect the number of fluorophores/chromogens that can be used in a single experiment. These include availability of microscope filter sets; such considerations are compounded when neurochemical identification uses combined FISH and IHC, compared to using each technique in isolation, since inherent steps optimal for one method may be detrimental to the other.
Previous application of FISH combined with IHC has demonstrated the expression of specific cellular targets in human B-cell lymphomas2, chick embryos3, zebrafish embryos4, mouse retina5 and mouse inner ear cells6. In these studies, tissue preparation was either formalin-fixed paraffin embedded (FFPE)2,3,5 or fresh whole mount4,6. Other studies applied chromogenic RNAscope on fixed mouse and rat brain preparations7,8,9. In particular, Baleriola et al.8 described two different tissue preparations for combined ISH-IHC; fixed mouse brain sections and FFPE human brain sections. In a recent publication, we combined FISH and fluorescent IHC on fresh frozen sections, to simultaneously visualize low abundance mRNA (galanin receptor 1, GalR1), high abundance mRNA (glycine transporter 2, GlyT2) and vesicular acetylcholine transporter (vAChT) protein10 in the brainstem reticular formation.
The nucleus of the solitary tract (NTS) is a major brain region involved in autonomic function. Located in the hindbrain, this heterogeneous population of neurons receives and integrates a vast number of autonomic signals, including those that regulate breathing. The NTS harbors several neuronal populations, which may be phenotypically characterized by the expression pattern of mRNA targets including GalR1 and GlyT2 and protein markers for the enzyme tyrosine hydroxylase (TH) and the transcription factor Paired-like homeobox 2b (Phox2b).
The RNAscope proprietor recommends fresh frozen tissue preparations, but tissue prepared by whole animal transcardial perfusion fixation, along with long term cryoprotection (storage at -20 &#176;C) of fixed frozen tissue sections, is common in many laboratories. Hence, we sought to establish protocols for FISH in combination with IHC using fresh frozen and fixed frozen tissue preparations. Here, we provide for fresh frozen and fixed frozen brain sections: (1) a protocol for combined FISH and fluorescent IHC (2) a description of the quality of mRNA and protein labelling produced, when utilizing each preparation (3) a description of the expression of GalR1 and GlyT2 in the NTS.
Our study revealed that, when combined with RNAscope methodology, IHC success varied in fresh frozen and fixed frozen preparations and, was dependent upon localization of the target proteins within the cell. In our hands, membrane bound protein labelling was always successful. In contrast, IHC for cytoplasmic protein required troubleshooting even in cases where the cytoplasmic protein was overexpressed in a transgenic animal (Phox2b-GFP)11. Finally, while GalR1 is expressed in non-catecholaminergic neurons in the NTS, GlyT2 expression is absent in the NTS.

<table><tbody><tr><td>&lt;strong&gt;ANIMALS&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>C57BL/6 mouse</td><td>Australian BioResources, Moss Vale</td><td>MGI: 2159769</td><td></td></tr><tr><td>Phox2b-eGFP mouse</td><td>Australian BioResources, Moss Vale</td><td>MGI: 5776545</td><td></td></tr><tr><td>&lt;strong&gt;REAGENTS&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Cyanoacrylate</td><td>Loctite</td><td></td><td></td></tr><tr><td>Ethylene Glycol</td><td>Sigma-Aldrich</td><td>324558</td><td></td></tr><tr><td>Heparin-Sodium</td><td>Clifford Hallam Healthcare</td><td>1070760</td><td>Consult local veterinary supplier or pharmacy.</td></tr><tr><td>Lethabarb (Sodium Pentabarbitol) Euthanasia Injection</td><td>Virbac (Australia) Pty Ltd</td><td>N/A</td><td>Consult a veterinarian for local pharmaceutical regulations regarding Sodium Pentabarbitol</td></tr><tr><td>Molecular grade agarose powder</td><td>Sigma Aldrich</td><td>5077</td><td></td></tr><tr><td>OCT Compound, 118mL</td><td>Scigen Ltd</td><td>4586</td><td></td></tr><tr><td>Paraformaldehyde, prilled, 95%</td><td>Sigma-Aldrich</td><td>441244-1KG</td><td></td></tr><tr><td>Polyvinylpyrrolidone, average mol wt 40,000&amp;nbsp; (PVP-40)</td><td>Sigma-Aldrich</td><td>PVP40</td><td></td></tr><tr><td>ProLong Gold Antifade Mountant</td><td>Invitrogen</td><td>P36930</td><td>With or without DAPI</td></tr><tr><td>RNAscope Multiplex Fluorescent Reagent Kit (up to 3-plex capability)</td><td>Advanced Cell Diagnostics, Inc. (ACD Bio)</td><td>ADV320850</td><td>Includes 50x Wash buffer and Protease III</td></tr><tr><td>RNase Away</td><td>Thermo-Fisher Scientific</td><td>7003</td><td></td></tr><tr><td>Tris(hydroxymethyl)aminomethane</td><td>Sigma-Aldrich</td><td>252859</td><td></td></tr><tr><td>Tween-20, for molecular biology</td><td>Sigma-Aldrich</td><td>P9416</td><td></td></tr><tr><td>&lt;strong&gt;EQUIPMENT&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Benchtop incubator</td><td>Thermoline scientific micro incubator</td><td>Model: TEI-13G</td><td></td></tr><tr><td>Brain Matrix, Mouse, 30g Adult, Coronal, 1mm</td><td>Ted Pella</td><td>15050</td><td></td></tr><tr><td>Cryostat</td><td>Leica</td><td>CM1950</td><td></td></tr><tr><td>Drawing-up needle (23 inch gauge)</td><td>BD</td><td>0288U07</td><td></td></tr><tr><td>Hydrophobic Barrier Pen</td><td>Vector labs</td><td>H-4000</td><td></td></tr><tr><td>Kimtech Science Kimwipes Delicate Task Wipes</td><td>Kimberley Clark Professional</td><td>34120</td><td></td></tr><tr><td>Olympus BX51</td><td>Olympus</td><td>BX-51</td><td></td></tr><tr><td>Peristaltic pump</td><td>Coleparmer Masterflex</td><td>L/S Series&amp;nbsp;</td><td></td></tr><tr><td>Retiga 2000R Digital Camera</td><td>QImaging</td><td>RET-2000R-F-CLR</td><td>colour camera</td></tr><tr><td>SuperFrost Plus Glass Slides (White)</td><td>Thermo-Fisher Scientific</td><td>4951PLUS4</td><td></td></tr><tr><td>Vibrating Microtome (Vibratome)</td><td>Leica</td><td>VT1200S</td><td></td></tr><tr><td>Whatman qualitative filter paper, Grade 1, 110 mm diameter</td><td>Merck</td><td>WHA1001110</td><td></td></tr><tr><td>&lt;strong&gt;SOFTWARES&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>CorelDRAW&amp;nbsp;</td><td>Corel Corporation</td><td>Version 7</td><td></td></tr><tr><td>FIJI (ImageJ Distribution)</td><td>Open Source/GNU General Public Licence (GPL)</td><td>N/A</td><td>ImageJ 2.x: Rueden, C. T.; Schindelin, J. &amp;amp; Hiner, M. C. et al. (2017), &quot;ImageJ2: ImageJ for the next generation of scientific image data&quot;, BMC Bioinformatics 18:529, PMID 29187165, doi:10.1186/s12859-017-1934-z&amp;nbsp;&amp;nbsp; and Fiji: Schindelin, J.; Arganda-Carreras, I. &amp;amp; Frise, E. et al. (2012), &quot;Fiji: an open-source platform for biological-image analysis&quot;, Nature methods 9(7): 676-682, PMID 22743772, doi:10.1038/nmeth.2019&amp;nbsp;</td></tr><tr><td>&lt;strong&gt;PRIMARY ANTIBODIES&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Anti-Tyrosine Hydroxylase Antibody</td><td>Millipore Sigma</td><td>AB1542</td><td>Sheep polyclonal (1:1000 dilution), RRID: AB_90755</td></tr><tr><td>Anti-Tyrosine Hydroxylase Antibody, clone LNC1</td><td>Millipore Sigma</td><td>MAB318</td><td>Mouse monoclonal (1:1000 dilution), RRID: AB_2201528</td></tr><tr><td>Anti-Vesicular Acetylcholine Transporter (VAchT) Antibody</td><td>Sigma-Aldrich</td><td>ABN100</td><td>Goat polyclonal (1:1000 dilution), RRID: AB_2630394</td></tr><tr><td>GFP Antibody</td><td>Novus Biologicals</td><td>NB600-308</td><td>Rabbit polyclonal (1:1000 dilution), RRID: AB_10003058</td></tr><tr><td>Phox2b Antibody (B-11)</td><td>Santa Cruz Biotechnology</td><td>sc-376997</td><td>Mouse monoclonal (1:1000 dilution), RRID: AB_2813765</td></tr><tr><td>&lt;strong&gt;SECONDARY ANTIBODIES&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Alexa Fluor 488 AffiniPure Donkey Anti-Rabbit IgG (H+L) (min X Bov, Ck, Gt, GP, Sy Hms, Hrs, Hu, Ms, Rat, Shp Sr Prot)&amp;nbsp;</td><td>Jackson ImmunoResearch</td><td>711-545-152</td><td>Donkey anti-Rabbit (1:400 dilution), RRID: AB_2313584</td></tr><tr><td>AMCA AffiniPure Donkey Anti-Sheep IgG (H+L) (min X Ck, GP, Sy Hms, Hrs, Hu, Ms, Rb, Rat Sr Prot)</td><td>Jackson ImmunoResearch</td><td>713-155-147</td><td>Donkey anti-Sheep (1:400 dilution), RRID: AB_AB_2340725</td></tr><tr><td>Cy5 AffiniPure Donkey Anti-Goat IgG (H+L) (min X Ck, GP, Sy Hms, Hrs, Hu, Ms, Rb, Rat Sr Prot)</td><td>Jackson ImmunoResearch</td><td>705-175-147</td><td>Donkey anti-Goat (1:400 dilution), RRID: AB_2340415</td></tr><tr><td>Cy5 AffiniPure Donkey Anti-Mouse IgG (H+L) (min X Bov, Ck, Gt, GP, Sy Hms, Hrs, Hu, Rb, Rat, Shp Sr Prot)</td><td>Jackson ImmunoResearch</td><td>715-175-151</td><td>Donkey anti-Mouse (1:400 dilution), RRID: AB_2619678</td></tr><tr><td>Cy5 AffiniPure Donkey Anti-Sheep IgG (H+L) (min X Ck, GP, Sy Hms, Hrs, Hu, Ms, Rb, Rat Sr Prot)</td><td>Jackson ImmunoResearch</td><td>713-175-147</td><td>Donkey anti-Sheep (1:400 dilution), RRID: AB_2340730</td></tr><tr><td>&lt;strong&gt;RNASCOPE PROBES&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Galanin Receptor 1 oligonucleotide probe</td><td>ACDBio</td><td>448821-C1</td><td>targets bp 482 - 1669 (Genebank ref: NM_008082.2)</td></tr><tr><td>Glycine transporter 2 oligonucleotide probe</td><td>ACDBio</td><td>409741-C3</td><td>targets bp 925 - 2153 (Genebank ref: NM_148931.3)</td></tr><tr><td>Phox2b oligonucleotide probe</td><td>ACDBio</td><td>407861-C2</td><td>targets bp 1617 - 2790 (Genebank ref: NM_008888.3)</td></tr></tbody></table>

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.

Here, we outline a method for combining multiplex FISH with fluorescent IHC to localize mRNA expression for GalR1 and GlyT2 using fresh-frozen and paraformaldehyde fixed tissues respectively in the mouse NTS. A pipeline of the tissue processing, FISH and IHC procedures described in the methods is displayed in Figure 1 and Figure 2. Table 1 provides a summary of the FISH probe and antibody combinations used in each figure.
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Watch this Scientific Journal Video about Combining Multiplex Fluorescence In Situ Hybridization with Fluorescent Immunohistochemistry on Fresh Frozen or Fixed Mouse Brain Sections at JoVE.com

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.

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 ...

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Neuroscience

6.8K Views.  University of New South Wales. 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.

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

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 ...

In Situ Immunohistochemical Detection of Immune Cell Markers in Cancer Tissues

Multiplex Cyclic Fluorescent Immunohistochemistry

The tumor microenvironment involves interactions between host cells, tumor cells, immune cells, stromal cells, and vasculature. Characterizing and spatially organizing immune cell subsets and target proteins are crucial for prognostic and therapeutic purposes. This has led to the development of multiplexed immunohistochemistry staining methods. Multiplex fluorescence immunohistochemistry allows the simultaneous detection of multiple markers, facilitating a comprehensive understanding of cell function and intercellular interactions. In this paper, we describe a workflow for the multiplex cyclic fluorescent immunohistochemistry assay and its application in the quantification analysis of lymphocyte subsets. The multiplex cyclic fluorescent immunohistochemistry staining follows similar steps and reagents as standard immunohistochemistry, involving antigen retrieval, cyclic antibody incubation, and staining on a formalin-fixed paraffin-embedded (FFPE) tissue slide. During the antigen-antibody reaction, a mixture of antibodies from different species is prepared. Conditions, such as antigen retrieval time and antibody concentration, are optimized and validated to increase the signal-to-noise ratio. This technique is reproducible and serves as a valuable tool for immunotherapy research and clinical applications.

Author Spotlight: Efficient Detection of Immune Cell-Infiltration in Cancer Tissues Using Fluorescent Immunohistochemistry

Multiplex cyclic immunohistochemistry allows in situ detection of multiple markers simultaneously using repeated antigen-antibody incubation, image scanning, and image alignment and integration. Here, we present the operating protocol for identifying immune cell substrates with this technology in lung cancer and paired brain metastasis samples.

The tumor microenvironment involves interactions between host cells, tumor cells, immune cells, stromal cells, and vasculature. Characterizing and ...

Cancer Research

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 ...

Immunofluorescence Staining Using IBA1 and TMEM119 for Microglial Density, Morphology and Peripheral Myeloid Cell Infiltration Analysis in Mouse Brain

This is a protocol for the dual visualization of microglia and infiltrating macrophages in mouse brain tissue. TMEM119 (which labels microglia selectively), when combined with IBA1 (which provides an exceptional visualization of their morphology), allows investigation of changes in density, distribution, and morphology. Quantifying these parameters is important in providing insights into the roles exerted by microglia, the resident macrophages of the brain. Under normal physiological conditions, microglia are regularly distributed in a mosaic-like pattern and present a small soma with ramified processes. Nevertheless, as a response to environmental factors (i.e., trauma, infection, disease, or injury), microglial density, distribution, and morphology are altered in various manners, depending on the insult. Additionally, the described double-staining method allows visualization of infiltrating macrophages in the brain based on their expression of IBA1 and without colocalization with TMEM119. This approach thus allows discrimination between microglia and infiltrating macrophages, which is required to provide functional insights into their distinct involvement in brain homeostasis across various contexts of health and disease. This protocol integrates the latest findings in neuroimmunology that pertain to the identification of selective markers. It also serves as a useful tool for both experienced neuroimmunologists and researchers seeking to integrate neuroimmunology into projects.

This protocol describes a step-by-step workflow for immunofluorescent costaining of IBA1 and TMEM119, in addition to analysis of microglial density, distribution, and morphology, as well as peripheral myeloid cell infiltration in mouse brain tissue.

This is a protocol for the dual visualization of microglia and infiltrating macrophages in mouse brain tissue. TMEM119 (which labels microglia ...

Immunology and Infection

A Rapid Method for Multispectral Fluorescence Imaging of Frozen Tissue Sections

Multispectral fluorescence imaging on formalin-fixed paraffin-embedded (FFPE) tissues enables the detection of multiple markers in a single tissue sample that can provide information about antigen coexpression and spatial distribution of the markers. However, a lack of suitable antibodies for formalin-fixed tissues may restrict the nature of markers that can be detected. In addition, the staining method is time-consuming. Here we describe a rapid method to perform multispectral fluorescence imaging on frozen tissues. The method includes the fluorophore combinations used, detailed steps for the staining of mouse and human frozen tissues, and the scanning, acquisition, and analysis procedures. For staining analysis, a commercially available semiautomated multispectral fluorescence imaging system is used. Through this method, up to six different markers were stained and detected in a single frozen tissue section. The machine learning analysis software can phenotype cells that can be used for quantitative analysis. The method described here for frozen tissues is useful for the detection of markers that cannot be detected in FFPE tissues or for which antibodies are not available for FFPE tissues.

We describe a rapid staining method to perform multispectral imaging on frozen tissues.

Multispectral fluorescence imaging on formalin-fixed paraffin-embedded (FFPE) tissues enables the detection of multiple markers in a single tissue sample ...

Free-floating Immunostaining of Mouse Brains

Immunohistochemical staining of mouse brains is a routine technique commonly used in neuroscience to investigate central mechanisms underlying the regulation of energy metabolism and other neurobiological processes. However, the quality, reliability, and reproducibility of brain histology results may vary among laboratories. For each staining experiment, it is necessary to optimize the key procedures based on differences in species, tissues, targeted proteins, and the working conditions of the reagents. This paper demonstrates a reliable workflow in detail, including intra-aortic perfusion, brain sectioning, free-floating immunostaining, tissue mounting, and imaging, which can be followed easily by researchers in this field.
Also discussed are how to modify these procedures to satisfy the individual needs of researchers. To illustrate the reliability and efficiency of this protocol, perineuronal nets were stained with biotin-labeled Wisteria florbunda agglutinin (WFA) and arginine vasopressin (AVP) with an anti-AVP antibody in the mouse brain. Finally, the critical details for the entire procedure have been addressed, and the advantages of this protocol compared to those of other protocols. Taken together, this paper presents an optimized protocol for free-floating immunostaining of mouse brain tissue. Following this protocol makes this process easier for both junior and senior scientists to improve the quality, reliability, and reproducibility of immunostaining studies.

This protocol describes an efficient and reproducible approach for mouse brain histological studies, including perfusion, brain sectioning, free-floating immunostaining, tissue mounting, and imaging.

Immunohistochemical staining of mouse brains is a routine technique commonly used in neuroscience to investigate central mechanisms underlying the ...

Indirect Immunofluorescence on Frozen Sections of Mouse Mammary Gland

Indirect immunofluorescence is used to detect and locate proteins of interest in a tissue. The protocol presented here describes a complete and simple method for the immune detection of proteins, the mouse lactating mammary gland being taken as an example. A protocol for the preparation of the tissue samples, especially concerning the dissection of mouse mammary gland, tissue fixation and frozen tissue sectioning, are detailed. A standard protocol to perform indirect immunofluorescence, including an optional antigen retrieval step, is also presented. The observation of the labeled tissue sections as well as image acquisition and post-treatments are also stated. This procedure gives a full overview, from the collection of animal tissue to the cellular localization of a protein. Although this general method can be applied to other tissue samples, it should be adapted to each tissue/primary antibody couple studied.

The indirect immunofluorescence protocol described in this article allows the detection and the localization of proteins in the mouse mammary gland. A complete method is given to prepare mammary gland samples, to perform immunohistochemistry, to image the tissue sections by fluorescence microscopy, and to reconstruct images.

Indirect immunofluorescence is used to detect and locate proteins of interest in a tissue. The protocol presented here describes a complete and simple ...

Biology

Histological-Based Stainings Using Free-Floating Tissue Sections

Immunohistochemistry is a widely used technique to visualize specific tissue structures as well as protein expression and localization. Two alternative approaches are widely used to handle the tissue sections during the staining procedure, one approach consists of mounting the sections directly on glass slides, while a second approach, the free-floating, allows for fixed sections to be maintained and stained while suspended in solution. Although slide-mounted and free-floating approaches may yield similar results, the free-floating technique allows for better antibody penetration and thus should be the method of choice when thicker sections are to be used for 3D reconstruction of the tissues, for example when the focus of the experiment is to gain information on dendritic and axonal projections in brain regions. In addition, since the sections are kept in solution, a single aliquot can easily accommodate 30 to 40 sections, handling of which is less laborious, particularly in large-scale biomedical studies. Here, we illustrate how to apply the free-floating method to fluorescent immunohistochemistry staining, with a major focus on brain sections. We will also discuss how the free-floating technique can easily be modified to fit the individual needs of researchers and adapted to other tissues as well as other histochemical-based stainings, such as hematoxylin and eosin and cresyl violet, as long as tissue samples are properly fixed, typically with paraformaldehyde or formalin.

The free-floating technique allows researchers to perform histological-based stainings including immunohistochemistry on fixed tissue sections to visualize biological structures, cell type, and protein expression and localization. This is an efficient and reliable histochemical technique that can be useful for investigating a multitude of tissues, such as brain, heart, and liver.

Immunohistochemistry is a widely used technique to visualize specific tissue structures as well as protein expression and localization. Two alternative ...

 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 ...

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

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