Name: combining multiplex fluorescence in situ hybridization with fluorescent immunohistochemistry on fresh frozen or fixed mouse brain sections
Uploaded: 2021-06-25T13:00:00
Description: 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

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...

<ol>
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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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>ANIMALS</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>REAGENTS</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&#160; (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>EQUIPMENT</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&#160;</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>SOFTWARES</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CorelDRAW&#160;</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. &#38; Hiner, M. C. et al. (2017), &#34;ImageJ2: ImageJ for the next generation of scientific image data&#34;, BMC Bioinformatics 18:529, PMID 29187165, doi:10.1186/s12859-017-1934-z&#160;&#160; and Fiji: Schindelin, J.; Arganda-Carreras, I. &#38; Frise, E. et al. (2012), &#34;Fiji: an open-source platform for biological-image analysis&#34;, Nature methods 9(7): 676-682, PMID 22743772, doi:10.1038/nmeth.2019&#160;</td>
		</tr>
		<tr>
			<td>PRIMARY ANTIBODIES</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>SECONDARY ANTIBODIES</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)&#160;</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>RNASCOPE PROBES</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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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 ...

This protocol combines fluorescence RNAscope in situ hybridization and immunohistochemistry using fresh-frozen or fixed brain preparations. This technique allows flexibility by demonstrating a combination of highly sensitive in situ hybridization and immunohistochemistry methods for spatial visualization of RNA and protein targets in the same tissue specimen. This method is applicable for multiplex labeling in brain tissue section, aiding the investigation of spatial organization and functional significance of mRNA and proteins within heterogeneous neuronal populations in neuroscience.This protocol mainly focuses on processing steps for fresh-frozen tissue and paraformaldehyde fixed tissue. To begin sectioning the fresh-frozen brain tissue, affix it to the pre-chilled cryostat chuck and cut 14 micrometer thick coronal sections. Mount the section onto a charged glass microscopy slide.Transfer the mounted slide into a slide box. Then transport the box to the minus 80 degree Celsius freezer on dry ice. For tissue fixation, filter the prepared paraformaldehyde solution.Cool it to 4 degrees Celsius. Bring the mounted slide from the minus 80 degree Celsius freezer on dry ice and immediately immerse it in the pre-chilled paraformaldehyde solution for 15 minutes, Perform dehydration of fresh-frozen tissue to ensure that the glass slide is completely dry. After removing the brain from a transcardially profusion fixed mount, using a vibrating microtome, cut 30 micrometer thick tissue sections.Store the cut sections in cryoprotectant solution. On the day of the FISH assay, transfer the free floating section into a 12-well cell culture plate containing 0.1 molar PBS and agitate it at 90 to 100 RPM on a rotating platform shaker to wash off cryoprotectant. Then, using a paintbrush, mount the section flat on a glass microscopy slide to prevent detachment during washes, and air dry for at least two hours.Using a hydrophobic barrier pen, draw a hydrophobic barrier around the section to contain the FISH reagents. Following pre-processing, the tissue is incubated in protease solution and hybridized with RNAscope probes for two hours as per the manufacturer's guidelines. Then, sequential amplification steps and fluorescence immunohistochemistry are performed.Start by preparing a humidified light protected chamber for incubating slides. In a water bath, warm 50X wash buffer and probes to 40 degrees Celsius for 10 minutes. Once cooled to room temperature, prepare one liter of 1X wash buffer from the 50X stock concentration.Prepare the probe mixture as described in the text manuscript. For proteinase treatment, add proteinase 3 on to the tissue section and incubate it at room temperature for 30 minutes. To wash off the proteinase, gently immerse the slide in 0.1 molar PBS at room temperature, avoiding section dislodging.For hybridization, add the probe mixture on to the tissue section. Place it in the humidified pre-warmed chamber. Then incubate it for two hours at 40 degrees Celsius.Rinse the tissue section with wash buffer twice for two minutes. For signal amplification, using a dropper bottle, add an amplification solution to cover the tissue section and incubate as described in the text. Add the blocking solution on to the tissue section and incubate for one hour at room temperature to prevent non-specific binding.Flick the slide to remove the excess blocking buffer after incubation. Now, incubate the section with primary antibodies overnight at 4 degrees Celsius. The next day, wash the slide three times with 1X TBS.Add the secondary antibody and incubate the sections for two hours at room temperature. Examine the slide under an epifluorescence microscope equipped with a camera. Acquire representative images at 20 times magnification and save them as TIF files.In the present study, the tissue quality, integrity, and the absence of bacterial contamination were confirmed by the absence of DAPI labeling. Labeling from positive control probes targeting ubiquitin C, peptidylprolyl isomerase B, and RNA polymerase IIA mRNA confirmed RNA integrity. In fresh-frozen preparations to distinguish GalR1 mRNA positive neurons within the NTS, additional neurochemical markers were used.The membrane-bound vesicular transporter was clearly labeled. In contrast, cytoplasmic proteins such as tyrosine hydroxylase and GFP expressed under the control of PHOX2B promoter were faintly observed and described as flocculent as they lack a clear outline. GalR1 FISH probe labeling of cytoplasmic GalR1 mRNA was punctate and clearly observed.To confirm that immunohistochemistry quality depends on protein subcellular localization, different nuclear and cytoplasmic proteins were labeled in parallel with FISH in the same tissue sections. The cytoplasmic PHOX2B GFP had a flocculent appearance. Whereas the nuclear PHOX2B protein signal was clear, which confirmed higher quality immunolabeling when combined with FISH.When performed on fixed frozen sections in combination with FISH, immunohistochemistry was reliable irrespective of subcellular localization. GlyT2 mRNA positive neurons were located ventral to the NTS and not within the NTS. GlyT2 mRNA positive and PHOX2B mRNA positive neurons did not colocalize.A subpopulation of PHOX2B mRNA positive NTS neurons was tyrosine hydroxylase immunoreactive, and none contained GlyT2 mRNA. RNAscope enables visualization and analysis of RNA distribution at single molecule resolution in cells and tissues. It empowers research in novel RNA species, disease mechanisms, gene expression patterns, neuron diversity, and cellular interactions.

Research

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Neuroscience

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