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

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.

Dispense the same number of drops of each DAB-A and DAB-B solution in an appropriately sized tube to make approximately 120 microliters of DAB substrate per section and vortex. Take each slide, one at a time, from the slide rack and tap and flick to remove excess liquid, and place it back in the slide rack. Pipette approximately 120 microliters of DAB mixture onto each tissue section, making sure that the sections are covered. Incubate for 10 minutes at room temperature and proceed with counterstaining.

Place one drop of mounting medium per glass slat and then place the slat on the slides and let them air dry. After a few hours, proceed with evaluation using an optical microscope as described in the manuscript.