I have stained MCF7 by DAPI quite OK using 0.5 ug/ml DAPI in PBS for 10 min. However, for A549, and HT29 I could not reach any staining result even by applying higher concentrations of DAPI (2ug/ml) and longer incubation time (30 min), or using permeablizer of Triton 0.2%. I have also checked the microscope function by previous samples and there was no error.
Any comments or suggestions?
Many thanks in advance.
Do you have an antifade reagent containing DAPI or HOESCHT? I have stained with DAPI in the past using the 2ug/ml and generally takes 5-30 minutes. I did this in C. elegans and in zebrafish embryos. I now use an antifade reagent that contains DAPI that adheres the coverslip to the slide. I now stain mouse tissue and smooth muscle cells and macrophages. Also, what is your fixation method?
Thanks for your answer dear Skylar King
I have not used any antifade reagent since the applied method worked quite fine in MCF7. The fixation method is incubation with formalin 4% in PBS, for 10 min prior to DAPI staining. I have also changed the DAPI source but it didn't work.
I have stained number of adherent cells including HepG2, Hepa1-6 and MCF7 with DAPI and Hoechst 33258 in early days of my doctorate. Protocol I used is as follows-
cells were fixed with 4% paraformaldehyde for 30 min at room temperature and washed twice with PBS. Chilled methanol (–20'C) was added to each well for 15 min at room temperature followed by 2–3 washes with PBS. Hoechst 33258 (10 mg/ml) was added for 10 min and washed with PBS and cells were examined by a fluorescent microscope (Nikon Eclipse 80i, Tokyo,Japan).
(FoxO proteins’ nuclear retention and BH3-only protein Bim induction evoke mitochondrial dysfunction-mediated apoptosis in berberine-treated HepG2 cells
S Shukla, F Rizvi, S Raisuddin, P Kakkar
Free Radical Biology and Medicine 76, 185-199)
Now, I use antifade reagent with DAPI. Both worked well with my cell lines @Fahimeh Zahednezhad
Are you protecting your DAPI from the light and diluting it from your stock fresh each time you use it?
This is highly unusual. For labeling fixed and permeabilized cells, I've never known DAPI to fail to label at that concentration and around a 5-10 minute label time, and I've used it in hundreds of different cell lines and tissue types. If there's dsDNA present, then it should label brightly.
The only things that will prevent labeling, in my experience, are 1) DNase contamination, 2) failure to permeabilize the sample beforehand, 3) co-labeling with a different nuclear stain which could cause FRET quenching, or 4) mounting in a mounting medium that quenches the signal (most aqueous mountants are fine, but some solvent-based mountants can cause quenching).
I'm right there with Jason A Kilgore: if you're following the manufacturer's protocol, DAPI should never fail. I've stained and imaged tens of mammalian cell lines, thousands of samples with differing fixation, permeabilisation, and staining protocols and have never seen DAPI or either of the Hoechst nuclear stains fail so long as the reagents were fresh.
Thank you all for sharing your valuable experiences.
I am wondering maybe some quenching problems has been occurred, still doubtful.
- Badges
- Science method