Extracellular DNA Staining: A Method to Identify Extracellular DNA in FFPE Tissue Sections
Trascrizione
A dying cell releases a fraction of DNA outside the cell called the extracellular DNA. To stain extracellular DNA, begin by taking a formalin-fixed paraffin-embedded, or FFPE, tissue section on a glass slide. Heat the glass slide for the desired duration. This facilitates the attachment of tissue on the slide surface and softens the surrounding paraffin.
Immerse the slide in deparaffinization liquid to completely dissolve the paraffin layer around the tissue. Next, dip the slide in decreasing concentrations of ethanol to rehydrate the deparaffinized tissue. Following rehydration, submerge the slide in boiling antigen retrieval solution. This treatment unmasks the epitopes that were altered during the fixation process.
Overlay the slide with primary antibodies and allow them to bind to specific unmasked epitopes present on the cell. Then, add fluorescently-tagged secondary antibodies, which bind to the primary antibodies. Dip the slide in Sudan Black solution to reduce background autofluorescence.
Finally, place a drop of mounting medium supplemented with DAPI over the tissue section and seal it with a coverslip. Incubate to allow the DAPI molecules to stain the DNA. Visualize the prepared tissue section using a fluorescence microscope. Fluorescently labeled epitopes in the cell help in differentiating the nuclear DNA present inside the cell from the extracellular DNA.
