Combining Multiplex Fluorescence In Situ Hybridization with Fluorescent Immunohistochemistry on Fresh Frozen or Fixed Mouse Brain Sections
Summary
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.
Abstract
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 "neurochemical signature" 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.
Introduction
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 °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.
Protocol
Representative Results
Discussion
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…
Acknowledgements
This work was funded by Australian Research Council Discovery Project grant DP180101890 and Rebecca L Cooper Medical Research Foundation project grant PG2018110
Materials
| ANIMALS | |||
| C57BL/6 mouse | Australian BioResources, Moss Vale | MGI: 2159769 | |
| Phox2b-eGFP mouse | Australian BioResources, Moss Vale | MGI: 5776545 | |
| REAGENTS | |||
| Cyanoacrylate | Loctite | ||
| Ethylene Glycol | Sigma-Aldrich | 324558 | |
| Heparin-Sodium | Clifford Hallam Healthcare | 1070760 | Consult local veterinary supplier or pharmacy. |
| Lethabarb (Sodium Pentabarbitol) Euthanasia Injection | Virbac (Australia) Pty Ltd | N/A | Consult a veterinarian for local pharmaceutical regulations regarding Sodium Pentabarbitol |
| Molecular grade agarose powder | Sigma Aldrich | 5077 | |
| OCT Compound, 118mL | Scigen Ltd | 4586 | |
| Paraformaldehyde, prilled, 95% | Sigma-Aldrich | 441244-1KG | |
| Polyvinylpyrrolidone, average mol wt 40,000 (PVP-40) | Sigma-Aldrich | PVP40 | |
| ProLong Gold Antifade Mountant | Invitrogen | P36930 | With or without DAPI |
| RNAscope Multiplex Fluorescent Reagent Kit (up to 3-plex capability) | Advanced Cell Diagnostics, Inc. (ACD Bio) | ADV320850 | Includes 50x Wash buffer and Protease III |
| RNase Away | Thermo-Fisher Scientific | 7003 | |
| Tris(hydroxymethyl)aminomethane | Sigma-Aldrich | 252859 | |
| Tween-20, for molecular biology | Sigma-Aldrich | P9416 | |
| EQUIPMENT | |||
| Benchtop incubator | Thermoline scientific micro incubator | Model: TEI-13G | |
| Brain Matrix, Mouse, 30g Adult, Coronal, 1mm | Ted Pella | 15050 | |
| Cryostat | Leica | CM1950 | |
| Drawing-up needle (23 inch gauge) | BD | 0288U07 | |
| Hydrophobic Barrier Pen | Vector labs | H-4000 | |
| Kimtech Science Kimwipes Delicate Task Wipes | Kimberley Clark Professional | 34120 | |
| Olympus BX51 | Olympus | BX-51 | |
| Peristaltic pump | Coleparmer Masterflex | L/S Series | |
| Retiga 2000R Digital Camera | QImaging | RET-2000R-F-CLR | colour camera |
| SuperFrost Plus Glass Slides (White) | Thermo-Fisher Scientific | 4951PLUS4 | |
| Vibrating Microtome (Vibratome) | Leica | VT1200S | |
| Whatman qualitative filter paper, Grade 1, 110 mm diameter | Merck | WHA1001110 | |
| SOFTWARES | |||
| CorelDRAW | Corel Corporation | Version 7 | |
| FIJI (ImageJ Distribution) | Open Source/GNU General Public Licence (GPL) | N/A | ImageJ 2.x: Rueden, C. T.; Schindelin, J. & Hiner, M. C. et al. (2017), "ImageJ2: ImageJ for the next generation of scientific image data", BMC Bioinformatics 18:529, PMID 29187165, doi:10.1186/s12859-017-1934-z and Fiji: Schindelin, J.; Arganda-Carreras, I. & Frise, E. et al. (2012), "Fiji: an open-source platform for biological-image analysis", Nature methods 9(7): 676-682, PMID 22743772, doi:10.1038/nmeth.2019 |
| PRIMARY ANTIBODIES | |||
| Anti-Tyrosine Hydroxylase Antibody | Millipore Sigma | AB1542 | Sheep polyclonal (1:1000 dilution), RRID: AB_90755 |
| Anti-Tyrosine Hydroxylase Antibody, clone LNC1 | Millipore Sigma | MAB318 | Mouse monoclonal (1:1000 dilution), RRID: AB_2201528 |
| Anti-Vesicular Acetylcholine Transporter (VAchT) Antibody | Sigma-Aldrich | ABN100 | Goat polyclonal (1:1000 dilution), RRID: AB_2630394 |
| GFP Antibody | Novus Biologicals | NB600-308 | Rabbit polyclonal (1:1000 dilution), RRID: AB_10003058 |
| Phox2b Antibody (B-11) | Santa Cruz Biotechnology | sc-376997 | Mouse monoclonal (1:1000 dilution), RRID: AB_2813765 |
| SECONDARY ANTIBODIES | |||
| 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) | Jackson ImmunoResearch | 711-545-152 | Donkey anti-Rabbit (1:400 dilution), RRID: AB_2313584 |
| AMCA AffiniPure Donkey Anti-Sheep IgG (H+L) (min X Ck, GP, Sy Hms, Hrs, Hu, Ms, Rb, Rat Sr Prot) | Jackson ImmunoResearch | 713-155-147 | Donkey anti-Sheep (1:400 dilution), RRID: AB_AB_2340725 |
| Cy5 AffiniPure Donkey Anti-Goat IgG (H+L) (min X Ck, GP, Sy Hms, Hrs, Hu, Ms, Rb, Rat Sr Prot) | Jackson ImmunoResearch | 705-175-147 | Donkey anti-Goat (1:400 dilution), RRID: AB_2340415 |
| Cy5 AffiniPure Donkey Anti-Mouse IgG (H+L) (min X Bov, Ck, Gt, GP, Sy Hms, Hrs, Hu, Rb, Rat, Shp Sr Prot) | Jackson ImmunoResearch | 715-175-151 | Donkey anti-Mouse (1:400 dilution), RRID: AB_2619678 |
| Cy5 AffiniPure Donkey Anti-Sheep IgG (H+L) (min X Ck, GP, Sy Hms, Hrs, Hu, Ms, Rb, Rat Sr Prot) | Jackson ImmunoResearch | 713-175-147 | Donkey anti-Sheep (1:400 dilution), RRID: AB_2340730 |
| RNASCOPE PROBES | |||
| Galanin Receptor 1 oligonucleotide probe | ACDBio | 448821-C1 | targets bp 482 – 1669 (Genebank ref: NM_008082.2) |
| Glycine transporter 2 oligonucleotide probe | ACDBio | 409741-C3 | targets bp 925 – 2153 (Genebank ref: NM_148931.3) |
| Phox2b oligonucleotide probe | ACDBio | 407861-C2 | targets bp 1617 – 2790 (Genebank ref: NM_008888.3) |
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