This video describes a sequential hybridization strategy that involves hybridizing mRNA probes to specific target mRNA. Following probe hybridization, signal amplification is performed to enhance resolution via reduction of signal-to-noise ratio during RNA-CISH of histological samples.
Protocol
All procedures involving human participants have been performed in compliance with the institutional, national and international guidelines for human welfare and have been reviewed by the local institutional review board. 1. Running the Assay NOTE: Do not let sections dry out between the incubation steps. Hybridization of the HPV probe NOTE: Ensure the probes are prewarmed to dissolve any precipit…
Disclosures
The authors have nothing to disclose.
Materials
HybEZ Oven (110v)
Advanced Cell Diagnostics Inc.
321710
HybEZ slide rack
Advanced Cell Diagnostics Inc.
300104
RNAscope 2.5 HD Detection Reagents-BROWN
Advanced Cell Diagnostics Inc.
322310
This kit includes amplification reagents AMP1, AMP2, AMP3, AMP4, AMP5 and AMP6, and detection reagents DAB-A and DAB-B
Probe Hybridization and Signal Amplification in RNA In Situ Hybridization: A Technique for Detecting Specific RNA Sequences in Tissue Sections. J. Vis. Exp. (Pending Publication), e20474, doi: (2023).