Chris,

The only requirement for getting fluorescein in solution is to
deprotonate it, i.e., making it alkaline. Just make a 50 mM stock
solution and pH it to about 8.0 with NaOH or other strong base and
keep it in the dark. You might try 10 uM for your patch concentration
but you can go to higher if you don't get a high enough signal. Doing
a calibration on a BioRad 1024, we got linear response from 0.5 uM up
to about 0.5 mM. The lower concentration requires accumulated frames,
but I think at 10 uM you're okay. 20 or 30 uM might be good, too,
more convenient even. Try and see.

I think I was able to get stock solutions up to 200 mM, but it is
more trouble and you don't need it.

Regards,
Mario

>I am going to put fluorescein in a patch pipette to visualize the
>neurons that I am patching.
>
>I know there is a trick to making solutions of fluorescein (raising the
>pH ?), but I don't have any experience. So if anyone with practical
>experience is listening..
>
>Questions:
>
>1) What would be a good concentration of dye in the patch pipette?
>
>2) Given the answer to question 1, how do I make a 1000x stock solution
>in water?
>
>3) Any other advice is welcome...
>
>PS I know that there are derivatives of Fluorescein that are easier to
>work with, but that is not an option at present.
>
>Thanks,
>
>
>Chris
>
>
>
>--
>Christopher Kushmerick  <[log in to unmask]>
>
>Dept. Farmacologia                              tel 55 31 499 2707
>Inst. Ciencias Biologicas                       fax 55 31 499 2695
>Universidade Federal Minas Gerais
>Av. Antonio Carlos 6627, Pampulha
>31270-901 Belo Horizonte, MG
>Brasil