Can Hoechst be used on live cells?
Hoechst and DAPI are popular blue fluorescent, nuclear-specific dyes that can be used to stain live or fixed cells. The dyes have minimal fluorescence in solution, but become brightly fluorescent upon binding to DNA. The staining is very stable and non-toxic to live cells for several days or longer.
Do you need to permeabilize cells for Hoechst staining?
Hoechst dyes are cell-permeable so there is no need to permeabilize them for Hoechst staining.
Can Hoechst 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.
Does Hoechst bind to RNA?
3.3 Hoechst 33258 fluoresces only about half as much when it binds to single-stranded genomic DNA compared to when it binds to double- stranded genomic DNA. 3.6 RNA does not interfere significantly with the DNA assay because Hoechst 33258 does not normally bind to RNA.
What does a Hoechst stain show?
Hoechst dyes are commonly used to stain genomic DNA in the following applications: Fluorescence microscopy and immunohistochemistry, often with other fluorophores. Flow cytometry to count or sort out cells.
How does Hoechst stain DNA?
Hoechst 33342 binds preferentially to adenine-thymine (A-T) regions of DNA. This stain binds into the minor groove of DNA and exhibits distinct fluorescence emission spectra that are dependent on dye:base pair ratios.
Is Hoechst membrane permeable?
Both DAPI and Hoechst are cell permeable. Unfortunately both require UV (or near UV) excitation so in any case they are not the best choice if you would like to image them in living cells.
What is the protocol for staining cells with Hoechst?
Protocol: Staining Cells with Hoechst or DAPI Nuclear Stains. Jump to a section: Hoechst and DAPI are popular blue fluorescent, nuclear-specific dyes that can be used to stain live or fixed cells. The dyes have minimal fluorescence in solution, but become brightly fluorescent upon binding to DNA.
How long does it take for Hoechst stain to work?
The optimal Hoechst-staining protocols are similar for multiple species. Wefound 90 minutes to be optimal for mouse SP cells, whereas 120 minutes is optimal forhuman, rhesus, and swine cells. Follow the protocol exactly as described above, butstain the bone marrow for 120 minutes.
How to prepare Hoechst staining solution for fluorescence microscope?
1. Culture cells in an appropriate medium and vessel for fluorescence microscopy. 2. Prepare the Hoechst staining solution by diluting the Hoechst® stock solution 1:2,000 in PBS. 3. Remove the medium. 4. Add sufficient staining solution to cover the cells.
What is the difference between DAPI and Hoechst staining?
In live yeast, Hoechst shows dim nuclear and cytoplasmic staining, while DAPI shows dim mitochondrial staining. The dyes can be used to stain yeast at 12-15 ug/mL in PBS.