We used the RNAscope Multiplex Fluorescent Reagent Kit v2 with the RNA-Protein Co-Detection Ancillary Kit for co-detection of mRNA and protein. For smFISH, we used custom-made RNAscope probes for Gad1, VGluT2 (Slc17a6), and NeuN (Rbfox3). Probes were based on the coding sequence of each gene, and single-nucleotide polymorphisms were included by alternating between species (P. maniculatus, P. polionotus). For IHC, we used the rabbit anti-c-Fos (1:100, Synaptic Systems, 226003) and rabbit anti-GFP (1:100, Thermo Fisher, A-11122) antibodies to detect c-Fos protein and the YFP tag in the viral vector, respectively, and HRP-labeled goat anti-rabbit antibody (1:500, PerkinElmer, NEF812001EA) for secondary detection. We visualized RNA probes and antibodies with Opal 520, Opal 570, and Opal 690 dyes (1:1000, Akoya Biosciences, FP1487001KT, FP1488001KT, FP1497001KT), and counterstained with DAPI. Regions of interest (mSC, dPAG) were imaged on a LSM 700 laser scanning confocal microscope (Zeiss), with Z-stacks of 21 slices spaced at 0.99 μm. We then used QuPath v0.2.3 to quantify the overlap of FISH and IHC signals in the maximum projection images.
Fp1488001kt
Manufactured by Akoya Biosciences
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FP1488001KT is a laboratory equipment product manufactured by Akoya Biosciences. It is designed to perform a core function, but a detailed description while maintaining an unbiased and factual approach is not available.
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Lab products found in correlation
Fp1487001kt, by Akoya Biosciences (5 mentions)
Fp1497001kt, by Akoya Biosciences (4 mentions)
A11122, by Thermo Fisher Scientific (1 mentions)
Lsm 700 confocal laser scanning microscope, by Zeiss (1 mentions)
Nef812001ea, by PerkinElmer (1 mentions)
Neurotrace 500 525 green fluorescent nissl stain, by Thermo Fisher Scientific (1 mentions)
Ultrapure dnase rnase free distilled water, by Thermo Fisher Scientific (1 mentions)
Bz 9000 all in one fluorescence microscope, by Keyence (1 mentions)
Vectra polaris, by Akoya Biosciences (1 mentions)
Bond 3, by Leica (1 mentions)
Opal 570, by Akoya Biosciences (1 mentions)
Opal 520, by Akoya Biosciences (1 mentions)
Opal 690, by Akoya Biosciences (1 mentions)
Rnascope multiplex fluorescent v2 assay, by Advanced Cell Diagnostics (1 mentions)
Fluoview fv1000, by Olympus (1 mentions)
Imaris, by Oxford Instruments (1 mentions)
Imaris software, by Oxford Instruments (1 mentions)
Fv3000 confocal microscope, by Olympus (1 mentions)
Rnascope multiplex fluorescent reagent kit v2, by Advanced Cell Diagnostics (1 mentions)
Ab208670, by Abcam (1 mentions)
9 protocols using fp1488001kt
1
Multiplex RNA-Protein Co-Detection in Brain
2
Fluorescent in situ Hybridization of Mouse Brain Tissue
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Mice were perfused with 3% PFA in UltraPure DNAse/RNAse-free distilled water (Cat. #10977015, Invitrogen). Brains were post-fixed overnight in 3% PFA, dehydrated through alcohols, and embedded in paraffin. Coronal sections at 6 μm were mounted onto Superfrost Plus slides. Sections were deparaffinized in xylene, washed in 100% ethanol, and dried at 65°C for 5 min. Then the sections were incubated with 100% hydrogen peroxide for 10 min, and washed 3 times. Sections next were steamed at 99°C for 15 min, encircled with a barrier pen, hybridized with a probe, and then incubated with amplifiers (Cat. #323136 and #447941, Advanced Cell Diagnostics). The fluorescence signal was developed using a fluorescent horseradish peroxidase substrate (Cat. #FP1488001KT, Akoya Biosciences). Following in situ hybridization, sections were counterstained with NeuroTrace 500/525 Green Fluorescent Nissl Stain (Cat. #N21480, ThermoFisher) and then sealed using Prolong Gold Antifade Mountant with DAPI. Images were acquired using a Keyence BZ-9000 All-in-one Fluorescence Microscope.
3
RNAscope Analysis of Murine Skin
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RNAscope in situ hybridization was performed using a Leica Bond III automated staining platform (Leica Biosystems) according to the manufacturer’s protocol. Mouse probes of Oca2 (ACDBio, 1072511), Gpr143 (ACDBio, 535108), Col12a1 (ACDBio, 312638) and Txnip (ACDBio, 457228) for the Leica System were used, and a RNAscope LS Multiplex Fluorescent assay (ACDBio) was used for detection. The double detection of immunofluorescence was performed after finishing in situ hybridization by applying the primary and secondary antibodies as described in the section ‘Immunofluorescence’. Skin sections were then scanned using Vectra Polaris (Akoya Biosciences) at ×20 for Opal fluors 570 (Akoya Biosciences, FP1488001KT), 690 (Akoya Biosciences, FP1497001KT) and DAPI (Akoya Biosciences, FP1490). Scanned images were processed using Inform (v.2.6.0) software (Aloya Biosciences). For quantitative analysis, HALO (v.3.5) software (module Indica Labs-FISH v.3.2.3) was utilized. FISH probe cell intensity (average intensity of FISH probe (×) spots and clusters per cell) was measured in tdTomato+ cells in the mid-anagen bulge/ORSup and the bulb. The relative intensity of bulge/ORSup tdTomato+ cells to bulb tdTomato+ cells within the same sample was compared across multiple samples.
4
Multicolor RNA Expression Analysis in Mouse Striatum
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Sagittal brain slices (∼12 μm thick) of WT mice were used to detect Drd1, Adora2a, and Fxyd2 gene expression in the striatum. RNAscope Multiplex Fluorescent Reagent version 2 (No. 323100; ACD) was used with dyes and dilutions as follows: 1:1500 Drd1 (high expression gene)-Opal520, 1:1500 Adora2a (high expression gene)-Opal570 and 1:750 Fxyd2 (low expression gene)-Opal690 (Nos. FP1487001KT, FP1488001KT, and FP1497001KT, respectively; Akoya Bioscience), following HybEZ II Hybridization System (No. 321721) protocols.
5
Ascl1 Expression Analysis via Fluorescent RNA-ISH
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We performed colorimetric RNA-in situ hybridization (ISH) using a digoxygenin-labeled Ascl1 riboprobe as previously described (Touahri et al., 2015 ). We performed fluorescent RNA-ISH using an RNAscope® Multiplex Fluorescent Detection Kit v2 (ACD #323110) and followed the manufacturer’s instructions. Briefly, brain sections were post-fixed (4% PFA/1XPBS) for 15 min at 4°C, and then, at room-temperature, dehydrated in 50%, 70%, and 100% ethanol (Commercial Alcohols, P016EAAN) for 5 min each, and incubated in H2O2 solution for 10 min. Sections were then incubated in 1x target retrieval solution for 5 min at 95°C, washed in dH2O, and then incubated in Protease Plus (ACD, 322331) for 15 min at 40°C before washing in washing buffer. We used a labeled RNA probe for Ascl1 (Mm-Ascl1 #313291) and used the negative and positive control probes provided. Sections were incubated with the probes for 2 h at 40°C. Amplification and staining steps were completed following the manufacturer’s instructions, using an Opal™ 570 (1:1500, Akoya #FP1488001KT) fluorophore.
6
In Situ Hybridization of TINAGL1 Expression
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In situ hybridization was performed using the RNAscope Multiplex Fluorescent V2 assay (Advanced Cell Diagnostics #323100), with probe for human TINAGL1 (Advanced Cell Diagnostics #857221-C2) and OPAL 570 fluorophore (Akoya #FP1488001KT), following the manufacturer’s protocol.
7
Visualization of Wnt6 mRNA in Corneal Tissue
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The RNAscope V2 Fluorescent Assay (ACD Biosystems, Newark, CA) was performed according to the ACD Biosystems protocol for fresh-frozen tissue. Corneal sections from 3 donors were hybridized with the Wnt6 mRNA probe. Assays were performed in 2 independent experiments for each donor. To confirm the mRNA integrity of the tissues, positive control probes targeting human housekeeping genes Polr2a, PPIB, and HPRT were visualized (Supplemental Fig. 1 ). The probes were amplified according to the manufacturer’s instructions and labeled with the fluorophore Opal 520 nm (Akoya Biosciences, FP1487001KT, 1:250), Opal 570 nm (Akoya Biosciences, FP1488001KT, 1:1500), and Opal 690 nm (Akoya Biosciences, SKU FP1497001KT, 1:1500). The Olympus FluoView FV1000 was used to visualize the fluorescence in situ hybridization signals. Quantitation of the dots among the limbal layers was performed with Imaris software and the surface and dots functions (V. 9.7.0, Imaris, Oxford Instruments, Oxon, UK).
8
Multicolor RNA Detection in Embryos
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RNAscope analysis was performed using RNAscope Multiplex Fluorescent Reagent Kit v2 (Advanced Cell Diagnostics #323100) with a few modifications to the previously described method by Nomaru et al. (50 (link)). Briefly, the embryos were fixed in 4% paraformaldehyde at 4 °C overnight. After dehydration and hydration in a graded series of methanol and PBS + 0.1% Tween, the embryos were treated with Protease III (#322102) for 20 min. The following C1, C2, and C3 probes were used for hybridization at 40 °C overnight: LacZ (#313451-C1), Nkx2-5 (#428241-C2), Tbx5 (#519581-C2), Tnnt2 (#418681-C3), Hand1 (#429651-C3), and Mab21l2 (#456901-C3). Following this, 0.2 × saline sodium citrate buffer (SSC) + 0.01% Tween was used for washing in between steps. The embryos were incubated with AMP1 for 30 min, AMP2 for 30 min, and AMP3 for 15 min at 40 °C. For signal detection, the embryos were incubated in HRP-C1, -C2, or -C3 for 15 min at 40 °C, followed by incubation in Opal 520, 690, and 570 (Akoya Biosciences #FP1487001KT, FP1497001KT, FP1488001KT). HRP was inactivated by HRP-Blocker for 15 min at 40 °C. After staining, the embryos were embedded in 50% glycerol/PBS. Fluorescent images were captured using an FV3000 confocal microscope (Olympus) with a Z-stack every 3 μm.
9
Multi-marker Immunofluorescence Profiling of Myocardial Samples
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Consecutive staining of 5 markers was performed on 3 µm sections of all myocardial samples on the same slide to enable colocalization assessment as described by Klinge et al. [22 (link)]. Immunofluorescence staining was carried out using primary antibodies for CD41 (ab203189, Abcam, Cambridge, UK), CD68 (ARP63008-P050; Aviva Systems Biology, San Diego, CA, USA), MPO (ab208670, Abcam, Cambridge, UK), and HIF2a (NB100-122, Novus Biologicals, Wiesbaden, Germany) according to a standard protocol. A secondary antibody, ImmPRESS® HRP Horse Anti-Rabbit IgG Polymer Detection Kit-Peroxidase (MP-7401; Vector Laboratories, Newark, NJ, USA), was used and coupled with an Opal dye, respectively. CD41 was labeled with Opal 480 (P1500001KT, Akoya Biosciences, Menlo Park, CA, USA), HIF2a with Opal 520 (FP1487001KT, Akoya Biosciences, Menlo Park, CA, USA), CD68 with Opal 570 (FP1488001KT, Akoya Biosciences, Menlo Park, CA, USA), and MPO with Opal 650 (FP1496001KT, Akoya Biosciences, Menlo Park, CA, USA). Hypoxyprobe™ was included as the fifth marker in this panel, as described below.
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