Can Hoechst stain fixed 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. Therefore, they can be used to stain cells without a wash step.
What is the purpose of fixing cells during DAPI staining?
It is believed that DAPI associates with the minor groove of double-stranded DNA, with a preference for the adenine-thymine clusters. Cells must be permeabilized and/or fixed for DAPI to enter the cell and to bind DNA. Fluorescence increases approximately 20-fold when DAPI is bound to double-stranded DNA.
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.
Does 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.
How does Hoechst stain work?
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.
What is the difference between Hoechst and DAPI?
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.
Is Hoechst DAPI?
Hoechst dyes are often used as substitutes for another nucleic acid stain called DAPI. Key differences between Hoechst dyes and DAPI are: Hoechst dyes are less toxic than DAPI, which ensures a higher viability of stained cells. The additional ethyl group of the Hoechst dyes renders them more cell-permeable.
Is Hoechst cell permeable?
Hoechst 33342 (2′-[4-ethoxyphenyl]-5-[4-methyl-1-piperazinyl]-2,5′-bi-1H-benzimidazole trihydrochloride trihydrate) is a cell-permeable DNA stain that is excited by ultraviolet light and emits blue fluorescence at 460 to 490 nm.
How to label cells with Hoechst stain?
Labeling cells 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. 5 Incubate for 5–10 minutes, protected from light.
How to prepare Hoechst stain for fluorescence microscopy?
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. 5. Incubate for 5–10 minutes, protected from light.
What is the Hoechst 33342 protocol?
Hoechst 33342 Protocol for Imaging Nuclear counterstain for fluorescence microscopy Invitrogen Hoechst 33342 nucleic acid stain is a popular cell-permeant nuclear counterstain that emits blue fluorescence when bound to dsDNA.
What are the advantages of Hoechst 33342 stain?
One advantage of Hoechst 33342 is that it is membrane permeant and, thus, can stain live cells. Hoechst 33342 binds to adenine-thymine-rich regions of DNA in the minor groove.