Hey!
I have a stock of 1:1000 DAPI. I used for 50 microliter of sample 1-2 microliter of DAPI and the stain is still very weak. I incubated DAPI with conjugated ab for 20 min, but even samples that waited for 1-1.5 hr for reading, still presented weak DAPI stain.
I know that my friends happen to use 1 to 20,000 with an incubation of 1 hr and didnt get across this issue.
Any ideas?
Dear Narin,
There may be many reasons for that, first ones coming to mind are as following: It may be that your cells are not very healthy, or depolarized and the nuclear membrane integrity is lost. If you are starting with cells with unhealthy nucleus the DAPI stain will look very weak. Other possibility is your imaging method and set up. Make sure you are using the right imaging with the correct excitation/emission with your microscopy. Perhaps you should compare your samples with your friends samples simultaneously and under the same conditions.
best wishes, Refik
To add to Refik's suggestions,
is your 1:1000 DAPI solution still a stock (ie high concentration) or is it already diluted, or is that your final concentration? ie, your vial of DAPI solution can have a too low concentration.
I use two types of Hoechst a lot, and always incubate 45-60 minutes. Hoechst for fixed samples is a final dilution of 1:1000, and for Hoechst for live experiments is 1:20.000
good luck
Hi, Narin,
Refik and Michiel both make good points, but I would add some others. First, separate your DAPI staining step from the other steps. DAPI staining can be your last step and staining shouldn't take more than five minutes! How old is your DAPI and how are you storing it? I usually store a 10mg/mL stock at -20 degrees C and dilute 30:1 for staining, so you may not be using enough stain. The buffer you use for washing your cells can have an effect as well. The protocol I use for staining can be found at http://olympus.magnet.fsu.edu/primer/techniques/confocal/applications/protocols/cellstriplebasic.html
http://olympus.magnet.fsu.edu/primer/techniques/confocal/applications/protocols/cellstriplebasic.html
Thank you all for your fine answers!
I work with fixated cells. After doubling or tripling the dose it was better. So i guess the reading is OK with the Imagestream device. Dapi was at 4* and was fine with live cells done by my fellow lab member. So i know it is OK. The Dapi was diluted by a fellow lab 1 to 1:1000 from the original stock.
When i place the DAPI with conjugated Ab for staining for 25 min i get quite a fine result, though as mentioned i know DAPI stain should be last final step and do not last more then 5-10 min! :-/
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