RNA CISH Signal Detection: A Technique to Detect Chromogenic Signals During RNA In Situ Hybridization in Intact Tissue Specimens
Transcription
To detect chromogenic signals during RNA hybridization, begin by taking a tissue section on a glass slide. This section contains target RNA, pre-hybridized with horseradish peroxidase-labeled RNA probe.
Dispense a few drops of diaminobenzidine or DAB, a detection substrate, over the tissue section and incubate.
DAB enters the cell, and the horseradish peroxidase enzyme oxidizes it to form a brown precipitate. This reaction imparts a brown color to specific RNA sequences in the cell. The cells that do not contain target RNA will have no peroxidase activity and remain unstained.
Next, counterstain the tissue section with hematoxylin. Hematoxylin binds to the DNA and imparts a purple stain to the tissue section.
Treat the tissue section with ammonia water – a bluing solution. It reduces the intensity of purple stain, making the cells appear blue. This step is essential for clear visualization of the brown spots inside the cells.
Now, immerse the tissue section in increasing concentrations of ethanol and subsequently treat it with xylene – an organic solvent. These steps dehydrate the tissue section to obtain better images.
Finally, seal the slide with a mounting solution and a coverslip and visualize under a light microscope. The cells exhibit punctate brown dots indicating the presence of target RNA.
