Sections were selected for the appropriate axial level, to include Rathke’s pouch or pituitary, as described previously (Lodge et al., 2016 (link)). The RNAscope 2.5 HD Reagent Kit-RED assay (Advanced Cell Diagnostics) was used with specific probes: Ctgf, Cyr61, Lats2 (all ACDBio).
Rnascope 2.5 hd reagent kit red assay
Manufactured by Advanced Cell Diagnostics
Sourced in United States
The RNAscope 2.5 HD Reagent Kit-RED assay is a highly sensitive in situ hybridization (ISH) technology that enables the detection and visualization of single-molecule RNA targets in fixed tissue samples. The kit provides the necessary reagents for performing the assay, including a probe, amplifiers, and chromogenic detection.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (4 mentions)
Alexa conjugated secondary antibody, by Thermo Fisher Scientific (2 mentions)
Fp1012, by PerkinElmer (2 mentions)
Rnascope fluorescent multiplex kit, by Advanced Cell Diagnostics (2 mentions)
Cm1850, by Leica (2 mentions)
Rnascope multiplex fluorescent v2 assay, by Advanced Cell Diagnostics (2 mentions)
Lats2, by Advanced Cell Diagnostics (1 mentions)
Rnascope fluorescent multiplex detection reagent kit, by Advanced Cell Diagnostics (1 mentions)
Hybez hybridization system, by Advanced Cell Diagnostics (1 mentions)
Rnascope multiplex fluorescent v2 kit, by Advanced Cell Diagnostics (1 mentions)
Axioscan, by Zeiss (1 mentions)
Brainmaker, by MBF Biosciences (1 mentions)
10 protocols using rnascope 2.5 hd reagent kit red assay
1
Rathke's Pouch RNA Expression Analysis
2
Embryonic Gene Expression Profiling
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Embryos harvested at predetermined stages were dissected and fixed in fresh 4% paraformaldehyde overnight at 4°C. Samples were passed through a sucrose series, embedded in OCT (optimal cutting temperature) compound (Tissue-Tek), and sectioned on a cryostat at 8 μm. Staining was performed using either an RNAscope 2.5 HD Reagent Kit-RED assay (322350, Advanced Cell Diagnostics) or an RNAscope Fluorescent Multiplex Detection Reagent kit (320851, Advanced Cell Diagnostics) according to the manufacturer’s instructions. Fgf8 (313411), Tbx1 (481911), Dlx5 (478151), Lhx6 (422791), Gsc (504821), Nbl1 (454541), Hand1 (429651), Alx1 (403161), Ebf1 (433411), Gbx2 (314351), Maf (412951), Osr2 (517481), Pitx1 (587451), Lix1 (552041-C2), Cgnl1 (587441), Spry2 (425061), and Cdk1 (476081-C2) probes were designed and synthesized by Advanced Cell Diagnostics.
3
Cryosectioning and Immunostaining of Decalcified Mandibles
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The decalcified mandibles were dehydrated in serial sucrose/PBS solutions and embedded in OCT compound (Tissue-Tek, Sakura). OCT-embedded samples were cryosectioned at 8 μm using a cryostat (Leica CM1850) followed by staining. For immunofluorescence staining, cryosections were soaked in blocking solution (PerkinElmer, FP1012) for one hour at room temperature and then incubated with primary antibodies diluted in blocking solution at 4°C overnight. After washing three times in PBS, the sections were incubated with alexa-conjugated secondary antibody (Thermo Fisher) and counterstained with DAPI (Thermo Fisher Scientific, 62248). The primary antibodies are listed in Table S1 . For in situ hybridization analysis, cryosections were stained with RNAscope Multiplex Fluorescent kit (Advanced Cell Diagnostics, 323100) or RNAscope 2.5 HD Reagent Kit-RED assay (Advanced Cell Diagnostics, 322350) according to the manufacturer’s instructions. All of the probes are listed in the Key resources table .
4
Cryosectioning and Immunostaining of Decalcified Mandibles
5
Dual-Staining of DAGLα and DAGLβ mRNA
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To detect and discriminate DAGLα and DAGLβ mRNA on cellular level, RNAscope® 2.5 HD Reagent Kit-RED assay (Advanced Cell Diagnostics, Cat#PN 322350) was used according to the manufacturer’s protocol. In short, 5 µm thick formaldehyde-fixed paraffin-embedded sections were de-paraffinized and pre-treated under standard pre-treatment conditions with hydrogen peroxide, target retrieval reagents and protease solution. The sections were covered with probe solution and incubated for 2 hours at 40°C using the HybEZ Hybridization System (Advanced Cell Diagnostics, Cat#PN 321710/321720). The sections were treated with AMP 1 to 6 according to the manufacturer´s manual, using the HybEZ Hybridization System. The multi-step hybridization process included hybridization to alkaline phosphatase-labeled probes and resulted in the detection of signal using Fast Red as a substrate. To combine ISH with immunohistochemistry (IHC), after performing ISH IHC was started from the blocking step as described below.
6
RNAscope Probe Evaluation for Gene Expression
7
RNAscope for FGF and DSPP detection
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Staining was performed on cryosections according to the manufacturer’s instructions using RNAscope 2.5 HD Reagent Kit-RED assay (Advanced Cell Diagnostics, 322350) or RNAscope Multiplex Fluorescent v2 kit (Advanced Cell Diagnostics, 323100). All probes used in this study were synthesized by Advanced Cell Diagnostics: Probe-Mm-Fgf1 (466661), Probe-Mm-Fgfr1 (454941), Probe-Mm-Fgfr2 (443501), Probe-Mm-Fgfr3 (440771), Probe-Mm-Fgfr4 (443511), Probe-Mm-Dspp (448301), and Probe-Mm-Gfra2-C2 (441481-C2).
8
RNAscope In Situ Hybridization Protocol
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RNAscope in situ hybridization in this study was performed on cryosections using RNAscope 2.5HD Reagent Kit-RED assay (Advanced Cell Diagnostics, 322350) and RNAscope multiplex fluorescent v2 assay (Advanced Cell Diagnostics, 323100) according to the manufacturer’s protocol. RNAscope probes used in this study included Kdm6a, Kdm6b, Uty, and Trp53. Detailed information about the probes is listed in Supplementary file 2 .
9
In situ Analysis of PACAP mRNA in Mouse Retina
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The eyeballs from three wild-type mice (C57BL/6N, n = 3) were freshly excised at each time point, frozen on Dry Ice, embedded in freezing medium (TissueTecTM) at −20°C and sectioned on a cryostat. Sagitta examine PACAP mRNA expression in retina by in situ histochemistry. The RNAscope 2.5 HD Reagent Kit-RED assay (Cat No. 322360, Advanced Cell Diagnostics, San Francisco, CA, United States) and mouse PACAP probe (Adcyap1, Cat No. 405911, Advanced Cell Diagnostics, San Francisco, CA, United States) were used for in situ hybridization of mouse retina sections following the user manual of the products. 50% hematoxylin staining solution was used for counterstaining. Slides with retina sections were image-captured with 20× objective with ZEISS Axio Scan (Carl Zeiss Microscopy, Thornwood, NY, United States) and images for sections from each animal were organized and converted to TIF files with BrainMaker (MBF Bioscience, Williston, VT, United States).
10
Multiplex RNAscope for Epigenetic Regulators
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RNAscope in situ hybridization in this study was performed on cryosections using RNAscope 2.5HD Reagent Kit-RED assay (Advanced Cell Diagnostics, 322350) and RNAscope multiplex fluorescent v2 assay (Advanced Cell Diagnostics, 323100) according to the manufacturer's protocol. RNAscope probes used in this study included Kdm6a, Kdm6b, Uty, and p53. Detailed information about probes is listed in Table S2.
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