the dilute concentration for DAPI staining - (Feb/05/2007 )

hello everyone/

I bought the DAPI from Sigma for contrastaining cell nuclei, acturally in the manip of immunocytostaining manipulation.

who could give me some advice about the dilute concentration for DAPI?
actually I also want use it as mounting solution, could somebody know I can do it or not, because I read many papers, they staining for 10 mins with DAPI staining, then rinse with PBS immediately. I want to know if I could let it on the samples and cover with cover slips??

My cells are MC3T3 E1 osteoblasts or L132 epithelial cells, after staining I will observe under confocal laser microscope.

thanks for attention

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If you plan to counter stain using DAPI separately, one does stain with DAPi then wash with PBS.

Or you could get a mounting media with DAPI (vector shield) so after the secondary antibody and washes, you directly mount the slides and neednot worry about DAPI.

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I agree with scolix - your best bet is the vector shield + dapi - i just put 10 ul on then slap on the coverslip - its foolproof

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thanks a lot to scolix and dominic/

But I am not really clear that if I should dilute the DAPI with mounting media or I just use DAPI staining and without rinsing afterwards and then put my mounting media on ?????

please could you explain clearer??? And for the dilute concentration, do you have some experience?

thanks again

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If the mounting media has DAPI (vector shield), you will have to apply the mounting media after the secondary antibody washes. No need to worry abt any dilution or washing away DAPI.

DAPI is toxic and a mutagen so its better to wash DAPI off. You will not go wrong with DAPI staining. I dont have the exact conc. for DAPI now.

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thanks a lot, I will try...

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Just to be sure you understand what we are saying
mounting medium from vectorshield is available with the dapi already added - i dont know anyone who uses dapi the same way you might use say toto3iodide where the dye would be incubated then washed off.

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thanks for reminding.

the whole story for me is like.

we use confocal laser macroscope to observe the stained cells, but DAPI acturally is not suitable for the wavelegnth of confocal laser macroscope , so we choose the TOPO3 as nuclear staining to get blue color, but TOPO 3 could not be observed by nake eyes in the observing lens, so we follow some advice to stain with DAPI too, so TOPO3 serve for the confocal laser macroscope to make digital image, DAPi serve for nake eye observation to aim at the interesting objectives.

Sorry for not tell the whole story, now you think it will be differnt or not. Sometimes I perhaps really think it too simple.

thanks and look forward to your confirmation

cherry

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interesting solution to another confusing lab situation.
using two nuclear stains is fine the only downside is it uses up wavelengths that could be put to better use elsewhere but unless youre doing some serious multistaining that wont be a problem.
have a peek at the vector website (online catalogue) and all your prayers will be answered (well at least for the next few hours)
good luck

dom

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thanks .

but could you directly give me the address of vector website ? Frankly speaking, I really know nothing about vector, it is the first time I hear. Hope you could make me know clearer about that. thanks again
lily

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