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.

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Neuroscience

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 ...

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Fluorescence Activated Cell Sorting (FACS) and Gene Expression Analysis of Fos-expressing Neurons from Fresh and Frozen Rat Brain Tissue

The study of neuroplasticity and molecular alterations in learned behaviors is switching from the study of whole brain regions to the study of specific sets of sparsely distributed activated neurons called neuronal ensembles that mediate learned associations. Fluorescence Activated Cell Sorting (FACS) has recently been optimized for adult rat brain tissue and allowed isolation of activated neurons using antibodies against the neuronal marker NeuN and Fos protein, a marker of strongly activated neurons. Until now, Fos-expressing neurons and other cell types were isolated from fresh tissue, which entailed long processing days and allowed very limited numbers of brain samples to be assessed after lengthy and complex behavioral procedures. Here we found that yields of Fos-expressing neurons and Fos mRNA from dorsal striatum were similar between freshly dissected tissue and tissue frozen at -80 &#186;C for 3 - 21 days. In addition, we confirmed the phenotype of the NeuN-positive and NeuN-negative sorted cells by assessing gene expression of neuronal (NeuN), astrocytic (GFAP), oligodendrocytic (Oligo2) and microgial (Iba1) markers, which indicates that frozen tissue can also be used for FACS isolation of glial cell types. Overall, it is possible to collect, dissect and freeze brain tissue for multiple FACS sessions. This maximizes the amount of data obtained from valuable animal subjects that have often undergone long and complex behavioral procedures.

Fluorescence Activated Cell Sorting FACS and Gene Expression Analysis of Fos-expressing Neurons from Fresh and Frozen Rat Brain Tissue

Here we present a Fluorescence Activated Cell Sorting (FACS) protocol to study molecular alterations in Fos-expressing neuronal ensembles from both fresh and frozen brain tissue. The use of frozen tissue allows FACS isolation of many brain areas over multiple sessions to maximize the use of valuable animal subjects.

The study of neuroplasticity and molecular alterations in learned behaviors is switching from the study of whole brain regions to the study of specific ...

Isolation of Cerebral Capillaries from Fresh Human Brain Tissue

Understanding blood-brain barrier function under physiological and pathophysiological conditions is critical for the development of new therapeutic strategies that hold the promise to enhance brain drug delivery, improve brain protection, and treat brain disorders. However, studying the human blood-brain barrier function is challenging. Thus, there is a critical need for appropriate models. In this regard, brain capillaries isolated from human brain tissue represent a unique tool to study barrier function as close to the human in vivo situation as possible. Here, we describe an optimized protocol to isolate capillaries from human brain tissue at a high yield and with consistent quality and purity. Capillaries are isolated from fresh human brain tissue using mechanical homogenization, density-gradient centrifugation, and filtration. After the isolation, the human brain capillaries can be used for various applications including leakage assays, live cell imaging, and immune-based assays to study protein expression and function, enzyme activity, or intracellular signaling. Isolated human brain capillaries are a unique model to elucidate the regulation of the human blood-brain barrier function. This model can provide insights into central nervous system (CNS) pathogenesis, which will help the development of therapeutic strategies for treating CNS disorders.

Isolated brain capillaries from human brain tissue can be used as a preclinical model to study barrier function under physiological and pathophysiological conditions. Here, we present an optimized protocol to isolate brain capillaries from fresh human brain tissue.

Understanding blood-brain barrier function under physiological and pathophysiological conditions is critical for the development of new therapeutic ...

Preparation of Mouse Retinal Cryo-sections for Immunohistochemistry

Preparation of high-quality mouse eye sections for immunohistochemistry (IHC) is critical for assessing the retinal structure and function and for determining the mechanisms underlying retinal diseases. Maintaining structural integrity throughout the tissue preparation is vital for obtaining reproducible retinal IHC data but can be challenging due to the fragility and complexity of retinal cytoarchitecture. Strong fixatives like 10% formalin or Bouin's solution optimally preserve the retinal structure, they often impede IHC analysis by enhancing the background fluorescence and/or diminishing antibody-epitope interactions, a process known as epitope masking. Milder fixatives, on the other hand, like 4% paraformaldehyde, reduces background fluorescence and epitope-masking, meticulous dissection techniques must be utilized to preserve the retinal structure. In this article, we present a comprehensive method to prepare mouse ocular posterior cups for IHC that is sufficient to preserve most antibody-epitope interactions without loss of retinal structural integrity. We include representative IHC with antibodies to various retinal cell type markers to illustrate tissue preservation and orientation under optimal and sub-optimal conditions. Our goal is to optimize IHC studies of the retina by providing a complete protocol from ocular posterior cup dissection to IHC.

This report describes comprehensive methods for preparing frozen mouse retina sections for immunohistochemistry (IHC). Methods described include dissection of the ocular posterior cup, paraformaldehyde fixation, embedding in Optimal Cutting Temperature (OCT) media and tissue orientation, sectioning and immunostaining.

Preparation of high-quality mouse eye sections for immunohistochemistry (IHC) is critical for assessing the retinal structure and function and for ...

Isolation of Adult Human Astrocyte Populations from Fresh-Frozen Cortex Using Fluorescence-Activated Nuclei Sorting

The complexity of human astrocytes remains poorly defined in primary human tissue, requiring better tools for their isolation and molecular characterization. Fluorescence-activated nuclei sorting (FANS) can be used to successfully isolate and study human neuronal nuclei (NeuN+) populations from frozen archival tissue, thereby avoiding problems associated with handling&#160;fresh tissue. However, efforts to similarly isolate astroglia from the non-neuronal (NeuN-) element are lacking. A recently developed and validated immunotagging strategy uses three transcription factor antibodies to simultaneously isolate enriched neuronal (NeuN+), astrocyte (paired box protein 6 (PAX6)+NeuN-), and oligodendrocyte progenitor (OLIG2+NeuN-) nuclei populations from non-diseased, fresh (unfixed) snap-frozen&#160;postmortem human temporal neocortex tissue.
This technique was shown to be useful for the characterization of cell type-specific transcriptome alterations in primary pathological epilepsy neocortex. Transcriptomic analyses confirmed that PAX6+NeuN- sorted populations are robustly enriched for pan-astrocyte markers and capture astrocytes in both resting and reactive conditions. This paper describes the FANS methodology for the isolation of astrocyte-enriched nuclei populations from fresh-frozen human cortex, including tissue dissociation into single-nucleus (sn) suspension; immunotagging of nuclei with anti-NeuN and anti-PAX6 fluorescently conjugated antibodies; FANS gating strategies and quality control metrics for optimizing sensitivity and specificity during sorting and for confirming astrocyte enrichment; and recommended procurement for downstream transcriptome and chromatin accessibility sequencing at bulk or sn resolution. This protocol is applicable for non-necrotic, fresh-frozen, human cortical specimens with various pathologies and recommended postmortem tissue collection within 24 h.

We have developed a method that enriches for and isolates human astrocyte populations from fresh-frozen tissue for use in downstream molecular analyses.