I have DAPI stock solution in DMSO (5 mg/ml) and need it for the use in immuno-fluorescence (IF) assay (mouse brain section). Generally we use 1:10,000 ration of DAPI with PBS in IF assay. At this condition, could you please let me know whether I should use stock solution directly (diluting in PBS at 1:10,000) or should I dilute it again before final dilution ( in PBS at 1:10,000) ?
Hi,
Unless you need 10 ml or more of the working solution, I'd recommend that you make a 1:10 dilution followed by a 1:1000 dilution in the final volume you require. Both can be made in PBS.
Best,
Anton
Hello,
I would say that you can prepare a 1:10,000 solution and store it at +4C up to 1 month.
The biggest amount I've stored this way was 50 ml in 1 single falcon.
Best
Hello..
I would suggest that you should dilute the DAPI stock solution, prepare its aliquots and store them at -20 degree C, before final dilution. At the time of use (IF), make it diluted and prepare final concentration.
Best wishes..
Hallo,
you can aliquot your stock solution and freeze it at -20°C as it is already mentioned. The used aliquot you can store in the fridge, do not freeze it again. You can dilute DAPI in the last rinsing buffer or directliy in the last anti-body solution.
After quite some attempts and vague references, we eventually found the correct dilution of DAPI from the powder.
We publish it in the Multiple Iteractive Labeling by Antibody Neodeposition (MILAN) protocol
https://www.nature.com/protocolexchange/protocols/7017
In part is derived from the CSHL protocol (http://cshprotocols.cshlp.org/content/2007/10/pdb.rec11127.full?text_only=true) and the key reagent is Methanol.
One may use DAPI dihydrochloride (D9542 Sigma), which comes in handy smaller and cheaper 1 mg aliquots that DAPI dilactate (Sigma D9564, 10 mg); this latter has the very same solubility problem, unless you use the procedure we published.
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