In situ hybridization was performed on 10 μm–thick coronal spinal cord sections using the RNAscope Multiplex Fluorescence V2 system (Advanced Cell Diagnostics) according to the manufacturer’s instructions. Twenty double-Z probes targeting Chat (catalog 408731) or small proline rich protein 1a (Sprr1a) (catalog 426871) mRNA were synthesized by Advanced Cell Diagnostics. Sections were deparaffinized and underwent multiple ethanol washes and peroxidase blocking steps. Sections were then incubated in antigen retrieval buffer and digested by proteinase. Sections were incubated in the double-Z probe mixture at 40°C in a hybridization oven for 2 hours. Sections were then washed, and the signal was amplified using the included amplification and HRP reagents. The sections were then imaged via confocal microscopy using identical imaging parameters across all samples. Maximum intensity projections were generated and used for analysis. We performed visual inspection of each Chat+ motor neuron in each tiled image for presence of Sprr1a signal.
Rnascope multiplex fluorescence v2 system
Manufactured by Advanced Cell Diagnostics
Sourced in United States
The RNAscope Multiplex Fluorescence V2 system is a tool for the simultaneous detection and visualization of multiple RNA targets within a single cell or tissue sample. It utilizes a proprietary signal amplification technology to enable the sensitive and specific detection of target RNA molecules.
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Lab products found in correlation
2 protocols using rnascope multiplex fluorescence v2 system
1
Spatiotemporal Analysis of Neuronal Molecular Profiles
2
Quantifying Epidermal Cell Proliferation
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The RNAScope® Multiplex fluorescence V2 system (Advanced Cell Diagnostics, Inc., Newark, CA, USA) was applied to in situ hybridization according to the manufacturer's instructions. Briefly, 10 μm paraffin skin sections from the AEW and Water groups were deparaffinized, rehydrated, washed in diethylpyrocarbonate (DEPC) distilled water, and stained with the following probes: mouse-Col17a1 (552141-C1; Advanced Cell Diagnostics) and mouse-Cux1 (442931-C2; Advanced Cell Diagnostics). After completing RNAScope, the skin sections were immediately subjected to Ki67 (ab15580, 1:250; Abcam, Waltham, MA, USA) immunofluorescence staining. The fluorescence intensities of Col17a1 and Cux1 in proliferating basal cells (stained positive for anti-Ki67 antibody in the epidermis) were quantified in both the AEW and Water groups. For skin sections from patients with psoriasis, we performed double immunofluorescence staining with Ki67 (ab15580, 1:250; Abcam) and Cux1 (sc-514008, 1:100; Santa Cruz Biotechnology, Inc., Dallas, TX, USA).
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