Hoechst 33342 Fluorescent Nucleic Acid Stain

1. Coat antibody or antigen onto the ELISA plate using ICT’s Antibody Coating Buffer or Antigen Coating Buffer., 2. Incubate 8–24 hours at room temperature (RT)., 3. Aspirate the coating solution., 4. Wash each Well twice with ICT’s ELISA Wash Buffer., 5. Block the uncoated regions of the ELISA plate by pipetting 300–400 µL of blocking buffer into each Well. Always use an equal or greater volume of blocking buffer than was used for the coating buffer solution., 6. Incubate 8–24 hours at RT. For best blocking, incubate overnight at RT., 7. Aspirate the blocking buffer; do not wash., 8. Run the assay immediately, or dry the plate for long-term storage and seal in a foil storage bag with a desiccant pack. Store dried and packaged plates at 2-8°C.

Hoechst 33342 is a popular cell-permeant, blue fluorescent nuclear stain. It is used to visualize the nuclei of living or fixed cells and tissues and is often used to distinguish condensed, pyknotic nuclei in apoptotic cells. Hoechst 33342 emits blue fluorescence when bound to double stranded DNA. It is slightly more membrane permeant than the Hoechst 33258 analog. Hoechst 33342 may be used to identify healthy or apoptotic nuclear morphology and for cell cycle studies.