FFPE Tissue Pretreatment for RNA CISH: A Procedure to Process Formalin-Fixed Paraffin-Embedded Tissue Sections for RNA Chromogenic In Situ Hybridization
Transcription
Pretreatment of formalin-fixed paraffin-embedded or FFPE tissue sections facilitates the exposure of nucleic acids and enables the binding of labeled probes to the target sequence during subsequent in situ hybridization. In addition, pretreatment helps maintain the integrity of the nucleic acids within the cells and overall tissue morphology.
To begin, take a glass slide carrying a deparaffinized and rehydrated FFPE tissue section of interest. Treat the tissue with an appropriate concentration of hydrogen peroxide and incubate.
Hydrogen peroxide irreversibly inactivates the endogenous peroxidase, which is physiologically present in the cells. This blocking process eliminates the possibility of non-specific background signals during subsequent hybridization steps.
Next, immerse the tissue section in a heated bath containing an appropriate antigen retrieval buffer. Incubate for the desired duration. High temperature cleaves fixative-induced chemical crosslinks in proteins and nucleic acids, exposing the unmasked epitopes to probes during subsequent labeling.
Finally, treat the specimen with a suitable protease solution. Incubate under a humidity-controlled environment. Proteases increase the accessibility of RNA, facilitating the specific probes to reach their target sequence in the following hybridization steps.
Wash the section with water to remove any residual proteases. Use the pretreated tissue section for RNA chromogenic in situ hybridization.
