Does Hoechst 33342 stain dead cells?
Yes, Hoechst 33342 can stain dead cells, however Hoechst 33358 is the preferred dye that’s used for staining dead or fixed cells. Hoechst 33342 is generally used for staining live cells. Hoechst dyes are a fluorescent stains that bind to AT-rich regions of the minor grove in DNA.
Can you stain live cells with Hoechst?
Hoechst and DAPI are popular blue fluorescent, nuclear-specific dyes that can be used to stain live or fixed cells. Both DAPI and Hoechst are minor-groove binding dyes with a preference for A/T-rich regions of DNA over G/C-rich DNA.
What is the fix solution used in the Hoechst DNA staining?
Properties of Hoechst Fluorescent Dye The stable 20 mM aqueous stock solution is essentially ready for use. Hoechst 33342 is used for specifically staining the nuclei of living or fixed cells and tissues.
How do you make a Hoechst stain?
Labeling cells
- Culture cells in an appropriate medium and vessel for fluorescence microscopy.
- Prepare the Hoechst staining solution by diluting the Hoechst stock solution 1:2,000 in PBS.
- Remove the medium.
- Add sufficient staining solution to cover the cells.
- Incubate for 5–10 minutes, protected from light.
How does Hoechst bind to DNA?
Hoechst 33342 binds to adenine-thymine-rich regions of DNA in the minor groove. On binding to DNA, the fluorescence greatly increases. This protocol describes the use of Hoechst 33342 to label nuclear DNA of cells grown in culture.
What is the difference between DAPI and Hoechst?
Hoechst dyes are typically used for staining DNA content in live cells due to its high cell membrane permeability. DAPI is typically used for staining DNA content in fixed cells due to its low membrane permeability.
Does Hoechst bind to RNA?
3.6 RNA does not interfere significantly with the DNA assay because Hoechst 33258 does not normally bind to RNA.
Do I need to Permeabilize for DAPI?
Sample preparation. Use the fixation protocol appropriate for your sample. DAPI staining is normally performed after all other staining. Note that fixation and permeabilization of the sample are not necessary for counterstaining with DAPI.