RNA CISH Signal Detection: A Technique to Detect Chromogenic Signals During RNA In Situ Hybridization in Intact Tissue Specimens

Abstract

Source: Outh-Gauer, S. et al. Chromogenic In Situ Hybridization as a Tool for HPV-Related Head and Neck Cancer Diagnosis. J. Vis. Exp. (2019)

This video presents the protocol for signal detection during RNA chromogenic in situ hybridization. This technique can be used to diagnose an active oncogenic infection visually in an intact tissue specimen.

Protocol

1. Preparation of Counterstaining Reagents

1. Prepare 50% hematoxylin.
   1. In a fume hood, add 100 mL of Gill’s hematoxylin I (see the Table of Materials) to 100 mL of distilled water in a staining dish.
      NOTE: The 50% hematoxylin staining solution can be reused for up to 1 week.
2. Prepare 0.02% (w/v) ammonia water (bluing reagent).
3. In the fume hood, add 1.43 mL of 1 N ammonium hydroxide to 250 mL of distilled water in a graduated cylinder or another container. Seal the cylinder with paraffin film. Mix its contents well for 3x–5x.
   ....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Hematoxylin solution, Gill No. 1	Merck	GHS132
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
RNAscope Probe- HPV16/18	Advanced Cell Diagnostics Inc.	311121
HybEZ slide rack	Advanced Cell Diagnostics Inc.	300104
ImmEdge Hydrophobic Barrier Pen	Advanced Cell Diagnostics Inc.	310018