Has the reagent itself allowed the entry of DAPI into the nucleus? I am using Annexin + PI staining and I could not permeabilize the cells previously. Thanks.
If tissues are fixed you should be able to use DAPI (Prolong Gold). We use it routinely in our preclinical and diagnostic IHC work.
A number of fluorescent stains are available that label DNA and allow easy visualization of the nucleus in interphase cells and chromosomes in mitotic cells, including Hoechst, 4′,6-diamidino-2-phenylindole (DAPI), ethidium bromide, propidium iodide, and acridine orange. Although not as bright as the vital Hoechst stains for DNA, DAPI has greater photostability. 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. This protocol describes the use of DAPI to label nuclear DNA of cells grown in culture.
Adapted from Imaging: A Laboratory Manual (ed. Yuste). CSHL Press, Cold Spring Harbor, NY, USA, 2010.
I normally use ProLong for mounting fixed samples (in propionic acid:formaldehyde:acetic acid) of ovules of Arabidopsis but stained with PI and works well.
Best wishes!
Yes, you can use it without permeabilizing the cells previously. DAPI can be used for alive cell but Hoechst doesn't.
If the cells are fixed then you do not really need to permeabilise them. DAPI is quite small and the holes in the cell membrane made by fixing will allow the dye in. I am not sure from your question whether you want to stain living cells, in which case I would not use it, but fixed cells are fine, and I have had great results using the prolong antifade mount ant.
I have used Prolong Gold Antifade w/ DAPI on paraformaldehyde-fixed frozen embryonic tissue cut to 7 microns thickness and find no difference in nuclear staining between permeabilizing and non-permeabilizing (detergent) conditions.
I have used Prolong Gold Antifade with DAPI on cytospun cells or frozen human skeletal muscle tissue sections, 5 µm thick, fixed with either acetone or formaldehyde w/ or w/o triton-x as permeabilizing agent, and I have had perfect results.
You don't need to permeabilise. works well in our sections too :)
I have used this same reagent just fixing the cells and worked fine.
Good luck with your experiments!
Thank you everybody!
I received the Invitrogen Scientific Assistance answer about this issue and they told me like is in the DAPI data sheet, “that fixation and permeabilization of the sample are not necessary for counterstaining with DAPI". I really didn’t know about that!
My best wishes for you!
Yes, I've done it several times and it works really well. You only have to fix the cells first.
i used this chemical pretty often with formalin-fixed paraffin section and acetone-fixed frozen section of peripheral nervous tissue on Superfrost™ Plus Gold Slides. Nucleus shall pick up the stain nicely!
best of luck
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