Three primary reagents used for these procedures include: HCL, proteinase treatment, and detergent treatment.
Protease treatment enhances accessibility to the target nucleic acid by breaking down the adjacent proteins. Typically, Proteinase K or pronase is used to remove those proteins.
Detergents are utilized to permeabilize cell membranes and disrupt lipid bilayers, thus facilitating the insertion of the hybridization probe into the tissue. Detergents like SDS and Triton X-100 are commonly used to permeabilize membranes by extracting lipid components.
HCl treatment is used to denature DNA and RNA molecules, making them more accessible for hybridization. By exposing the tissue to acidic conditions, HCl disrupts the hydrogen bonds between the nucleotide base pairs. This treatment is believed to contribute to a reduction in background staining by hydrolyzing the target sequence and extracting proteins.