This protocol describes a method for combining fluorescence in situ hybridization (FISH) and fluorescence immunohistochemistry (IHC) in both fresh frozen and fixed mouse brain sections, with the goal of achieving multilabel FISH and fluorescence IHC signal. IHC targeted cytoplasmic and membrane attached proteins.
Fluorescent in situ hybridization (FISH) is a molecular technique that identifies the presence and spatial distribution of specific RNA transcripts within 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.
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.
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
2. Sample preparation of fixed frozen brain tissue
3. FISH assay
NOTE: The rest of the protocol applies to both fresh frozen and fixed frozen tissue.
4. IHC Assay
5. Imaging
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.
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. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
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.
Control probes are routinely assayed concurrently with target probe, to ensure integrity of the workflow and confirm sample quality. The absence of DapB labelling confirms sound tissue quality and integrity, and the absence of bacterial contamination (Figure 3A). Labelling from positive control probes targeting ubiquitin C (UBC, high abundance), peptidylpropyl isomerase B (PPIB, moderate abundance) and RNA polymerase 2a (POLR2A, low abundance) mRNA confirms RNA integrity and the signal observed between assays may be used to calibrate inter assay variability (Figure 3B). To validate FISH probe expression, we used control tissues that have been previously described to express the mRNA transcript. For example, GalR1 mRNA expression, was confirmed to be positive in the thalamus as previously described10,15. Phox2b mRNA distribution was additionally verified by colabelling with Phox2b antibody; we confirmed that FISH labelling was present only in neurons that were also positively stained using the Phox2b antibody (Figure 5).
To distinguish GalR1+ neurons in the NTS from neighbouring nuclei, we used additional neurochemical markers. TH, Phox2b or Phox2b-GFP immunoreactivity (Figure 4-6), and Phox2b FISH (Figure 5 and Figure 6) differentiated the NTS from other nuclei in the dorsal brainstem as NTS neurons have previously been reported to express Phox2b and TH16,17. Since the NTS is nestled by cholinergic nuclei – it lies dorsal to the hypoglossal nucleus and dorsal motor nucleus of the vagus (DMNX), and ventral to the vestibular nucleus – we co-labelled with the cholinergic marker vAChT18 (Figure 4). Therefore, the expression of GalR1 within the NTS was assessed in relation to TH and Phox2b, whilst vAChT labelling aided spatial orientation with respect to rostrocaudal, dorsoventral and mediolateral coordinates. We found all TH immunoreactive and GalR1 mRNA positive neurons in the NTS were Phox2b-GFP immunoreactive, but not all Phox2b-GFP immunoreactive neurons in the NTS were TH immunoreactive or GalR1 mRNA positive (Figure 4). Also, we demonstrated that mRNA for the low abundance receptor GalR1 was absent in TH and vAChT immunoreactive neurons.
In fresh frozen preparations, when combined with the FISH probe assay, IHC success was dependent on subcellular location of the target protein. For example, vAChT (a synaptic vesicle membrane-bound protein) was clearly immunolabelled, whereas TH and GFP (cytoplasmic proteins) were indefinitely immunolabelled and only faintly observed (Figure 4). We describe this indefinite labelling as 'flocculent' because cells lacked a clear outline and proved difficult to distinguish from the background. On the same fresh frozen tissue section, GalR1 FISH probe labelling of cytoplasmic GalR1 mRNA was punctate and clearly observed (Figure 4).
Furthermore, since the TH and vAChT antibodies are raised in the same host, both proteins were labelled using the same secondary antibody and therefore the same color fluorophore (excitation light: 594). They are easily distinguished for two reasons: they never co-label in the same neurons, and the subcellular localisation is different for these proteins; vAChT in vesicles exhibiting a punctate appearance, and TH in the cytoplasm and neuronal processes.
To support our hypothesis that IHC quality (in fresh frozen preparations) is dependent on protein subcellular localisation, we compared labelling for Phox2b mRNA (located in the cytoplasm), GFP (over-expressed in cytoplasm) and Phox2b protein (primarily found in the nucleus) in neurons. As expected, our results show overlap of Phox2b mRNA, GFP and Phox2b antibody labelling in individual neurons of the NTS (Figure 5). Cells with cytoplasmic mRNA labelling corresponded with cells exhibiting nuclear labelling of the Phox2b protein providing validation of the combined FISH-IHC method. Although cytoplasmic Phox2b-GFP had a flocculent appearance, nuclear Phox2b protein signal was clear and specific. In conclusion, when combined with FISH on fresh frozen preparations, membrane-bound proteins including vAChT and Phox2B exhibit higher quality immunolabelling than cytoplasmic proteins.
In contrast, IHC was reliable irrespective of subcellular localization, when performed on fixed frozen sections in combination with FISH. Multiplex FISH for GlyT2 mRNA and Phox2b mRNA was successful, as shown in Figure 6. GlyT2 mRNA positive neurons were located ventral to the NTS and not within the NTS. GlyT2+ and Phox2b+ neurons did not colocalize. A subpopulation of Phox2b+ NTS neurons was TH immunoreactive and none contained GlyT2 mRNA. TH immunoreactive neurons are apparent on the same tissue section, exhibiting positively labelled soma and neuronal processes (Figure 6). This contrasts with the 'flocculent' appearance of TH immunoreactive neurons in fresh frozen tissue sections. Thus, the fixed frozen preparation described here is an alternative method of tissue preparation which enables reliable targeting of cytoplasmic proteins immunohistochemically, in combination with RNAscope.
Figure 3: Representative microscopic images from coronal mouse forebrain sections at the level of the lateral septum (Bregma 1.1 to -0.1) showing labelling of positive and negative control probes. (A) A lack of signal following ISH with bacterial 4-hydroxy-tetrahydrodipicolinate reductase (DapB) confirms the absence of background signals. (B) Labelling with positive control probes targeting ubiquitin C (UBC), peptidylpropyl isomerase B (PPIB) and RNA polymerase 2a (POLR2A) illustrates the signal to be expected from high, moderate and low abundance targets respectively. Scale bars are 50 µm. All images were acquired with 20x objective. Please click here to view a larger version of this figure.
Figure 4: Representative microscopic images of a fresh frozen coronal brainstem section from a Phox2b-GFP mouse showing combined labelling of GalR1 mRNA (FISH) and 3 proteins (IHC) in the nucleus of the solitary tract (NTS) region. Insets in A are enlarged in B. GalR1 mRNA is indicated by punctate FISH probe labelling (arrowheads). Antibodies targeting the cytoplasmic proteins GFP and tyrosine hydroxylase (TH) exhibited "flocculent" labelling (arrows). Vesicular acetylcholine transporter (vAChT) immunoreactivity is demonstrated (red punctate labelling) in the hypoglossal nucleus (XII). Scale bars are 100 µm in A and 25 µm in B. All images were acquired with 20x objective. Other abbreviations: area postrema (AP), medial vestibular nucleus (MVe). Please click here to view a larger version of this figure.
Figure 5: Representative microscopic images of a fresh frozen coronal brainstem section from a Phox2b-GFP mouse, illustrating targeting of Phox2b in the nucleus of the solitary tract (NTS) with three different approaches: Phox2b mRNA (FISH), GFP (IHC) and Phox2b protein (IHC). Phox2b protein is localized to the nucleus. Insets in A are enlarged in B. Arrows indicate neurons that are triple labelled with Phox2b probe (orange-550), GFP antibody (green-488) and Phox2b antibody (red-647). Scale bars are 100 µm in A and 25 µm in B. All images are acquired with a 20x objective. Other abbreviations: area postrema (AP). Please click here to view a larger version of this figure.
Figure 6: Representative images from fixed frozen coronal brainstem sections demonstrating successful FISH combined with reliable immunolabelling of cytoplasmic proteins (tyrosine hydroxylase [TH]). Double FISH showing glycine transporter 2 (GlyT2-red-647, filled arrowheads) and Phox2b (yellow-550, arrows) mRNA labelling in nucleus of the solitary tract (NTS) region. FISH was combined with IHC for TH protein (blue-346, empty arrowheads). Insets in A are enlarged in B. Scale bars are 25 µm. All images were acquired with a 20x objective. Please click here to view a larger version of this figure.
Primary antibody or RNAscope probe | Secondary antibody or Amp 4-FL-Alt Display module |
Excitation (nm) | Tissue preparation | ||
Figure 3 | probe | POLR2A (C1) | Amp 4-FL-Alt B Display module | 647 | fresh frozen |
probe | PPIB (C2) | Amp 4-FL-Alt B Display module | 488 | ||
probe | UBC (C3) | Amp 4-FL-Alt B Display module | 550 | ||
probe | DapB (C1, C2, C3) | Amp 4-FL-Alt B Display module | 647, 488, 550 | ||
DAPI | 346 | ||||
Figure 4 | antibody | rabbit-anti-GFP | donkey-anti-rabbit | 488 | fresh frozen |
antibody | sheep-anti-TH | donkey-anti-sheep | 647 | ||
antibody | goat-anti-vAChT | donkey-anti-goat | 647 | ||
probe | GalR1 (C1) | Amp 4-FL-Alt B Display module | 550 | ||
DAPI | 346 | ||||
Figure 5 | antibody | rabbit-anti-GFP | donkey-anti-rabbit | 488 | fresh frozen |
antibody | mouse-anti-Phox2b | donkey-anti-mouse | 647 | ||
probe | Phox2b (C2) | Amp 4-FL-Alt A Display module | 550 | ||
DAPI | 346 | ||||
Figure 6 | antibody | mouse-anti-TH | donkey-anti-mouse | 346 | fixed |
probe | GlyT2 | Amp 4-FL-Alt A Display module | 647 | ||
probe | Phox2b | Amp 4-FL-Alt A Display module | 550 |
Table 1: FISH probe, antibody and corresponding flurophore combinations used in Figures 3-6.
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 reliable IHC outcomes. For example, multiplex FISH for low and high abundance mRNAs (GalR1 and GlyT2 respectively) and IHC (targeting tyrosine hydroxylase) revealed that GalR1 and GlyT2 are expressed in non-catecholaminergic NTS neurons. IHC for TH was not successful in fresh frozen tissue, highlighting the limited capacity for FISH-IHC in fresh frozen preparations.
ISH may be more appropriate than IHC in a range of scenarios. First, IHC may not perform well when detecting low abundance proteins, such as receptors. Using ISH to target relatively higher abundance mRNAs for these proteins improves detectability1. Second, proteins such as neuropeptides are often trafficked to the axonal terminals following translation in the cell soma19. When neuropeptides are targeted with IHC, the axonal processes and terminals of cells label with the antibody, but not the soma, reducing the capacity to identify the cell of origin or perform quantitative analysis of the number of cells. However, since mRNAs which code for all proteins are found localized to the soma, the ISH technique is advantageous. Finally, antibodies are not readily available for some protein species, or the available antibodies are appropriate for other proteomics techniques (e.g., western blot) but not IHC. In these circumstances, mRNA labelling methods prove useful. A caveat is that mRNAs may not always be translated into protein, and so they only provide a proxy for protein identification. Since commercial FISH kits can be costly and the ISH probes are less likely to be commercially available compared to antibodies, combining FISH with IHC presents a cost and time effective strategy for increasing the number of targets that can be labelled simultaneously.
Fresh frozen versus fixed tissue preparation was a factor conferring successful IHC following FISH probe assay. We tested IHC using antibodies targeting nuclear, vesicular and cytoplasmic proteins and found reliable labelling of membrane-bound proteins (vAChT and Phox2b) on fresh frozen samples but not cytoplasmic proteins (TH and GFP). Coexpression of Phox2b protein and mRNA with 'flocculent' Phox2b-GFP labelling validated that neurons expressing the transcript also expressed the related protein, confirming the neurochemical identity of the neurons (Figure 5). In contrast, fixed frozen tissue preparations yielded reliable IHC labelling regardless of subcellular localization of the antigen. Previous studies have demonstrated that protease (e.g., pronase8,20) pretreatment can have a detrimental effect on IHC. The contents of the protease solution utilized in the RNAscope protocol is proprietary, and permeabilization by protease is recommended for RNAscope probe access into cells. Labelling of cytoplasmic proteins using the antibodies described here has been previously verified on free floating 30 µm fixed frozen mouse brain sections10,21,22. We slide-mounted 30 µm thick fixed samples and performed the FISH-IHC protocol, as opposed to the 14 µm thick fresh frozen sections recommended by the manufacturer. In the absence of assay modifications, or alteration of other variables (antigen retrieval, higher antibody concentration, change of protease), reliable IHC was achieved on thick, fixed samples with demonstrated labelling of the cytoplasm and axonal processes together with FISH probe labelling (Figure 6). While similar approaches were employed by other research groups7,8,9, the current study achieved combined ISH-IHC on neurons and in a fluorescent set-up.
There were a series of critical steps in the methods to take note of. For the fresh frozen preparation, fixation time should not exceed 15 minutes; longer fixation times elicited higher background labelling. The protease step was optimized since tissues of different thickness and from various organs require different types of protease to achieve permeabilization. Fixed frozen sections adhere less to glass slides and dislodge more easily during wash steps. Hence, extra care must be taken in manual handling of fixed frozen sections, to avoid tissue loss or damage.
Although we found combined FISH and IHC to be an effective strategy, the disadvantages include cost and technically demanding assay when combining the two methods. One limitation of the study is that a side-by-side comparison of the two tissue preparation protocols was not performed. Also, our evaluation of results was limited by the number of channels the epifluorescent microscope could accommodate; the set-up allowed a maximum of 4 channels at a given time: 346, 488, 550 and 647 nm (excitation light). We were able to achieve multiplex labelling of 5 targets by labelling two proteins with different subcellular localizations using the same flurophore (Figure 4, Table 1). By using a confocal microscope, discrete excitation of many additional fluorophores may be used for individual protein labelling via IHC, or for imaging of fluorescent molecules expressed by transgenes.
Combined FISH and fluorescent IHC can reduce the reliability of each technique in isolation. In the future, we aim to improve cytoplasmic protein labelling on fresh frozen tissue with an antigen retrieval treatment23. Previous studies show that heat induced antigen retrieval increases accessibility of the protein epitope24,25,26. Heat treatment cleaves the crosslinks and methylol groups of the protein and unfolds the antigens in tissues, exposing epitopes which would otherwise be hidden in the tertiary protein structure under biological conditions. This accessibility may improve the success of protein labelling26,27. Additionally, we will target different epitopes of the same cytoplasmic protein to determine if the success of protein-antibody labelling depends on the specific antibody clones used.
In conclusion, combined FISH and IHC is useful for neurochemical identification of heterogenous populations of cells in the brain, such as those in the NTS. This study presents two protocols assaying different mouse brainstem tissue preparations – fresh frozen, or fixed – for simultaneous multiplex fluorescent labelling of mRNA and proteins in situ. Both protocols may be widely applied to detect the expression pattern of low abundance mRNAs, such as GalR1. Thick (30 µm) fixed frozen preparations permeabilized with protease conferred more reliable cytoplasmic protein detection and more tissue handling challenges, when compared to thin (14 µm) fresh frozen preparations.
This work was funded by Australian Research Council Discovery Project grant DP180101890 and Rebecca L Cooper Medical Research Foundation project grant PG2018110
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) |