Re: Ca-measurement in glandular tissue

Posted by Axel Kurt Preuss on May 11, 2010; 10:05am
URL: http://confocal-microscopy-list.275.s1.nabble.com/Ca-measurement-in-glandular-tissue-tp5035189p5035232.html

Dear Attila
1.5 hrs is far too long
I would try 40 min, maybe even only 30 min
Then washing in HBSS. The phosphate in PBS can be a problem (catching
your Ca away)
I would clamp Ca down to 1 mM , maybe even 300 micro M during washing

But the most critical step is what cells you want to label. If you
soak the whole tissue in fluo 4 you might get abundant fluorescence
making cells indiscriminable or buffer too much Ca



Cheers

Axel

Axel K Preuss PhD, A*Star IMCB-Central Imaging Facility 6-19B   Sent
from. [phone]

On May 11, 2010, at 5:50 PM, "Cselenyak Attila" <[hidden email]>
wrote:

> Dear List members,
>
> we would like measure Ca(2+) in glandular tissue with Fluo3 using
> confocal mircoscopy. We tried to load the tissue pieces with different
> concentrations of the dye for 1,5 hours in 37C, washing after that
> with PBS, and the tissue was put in a perfusion system. But we could
> not see any changes in the fluorescence intensity when giving either
> ATP or A23187 ionophore to the perfusion system.
>
> Do you have any experience what would be an optimal loading protocol
> or Fluo3 is suitable for this kind of measurements?
>
> Thanks is advence!
>
> Best regards,
>
> Attila
>
> --
> Attila Cselenyak
> PhD student
>
> Semmelweis University
> Basic Medical Science Center
> Institute Of Human Physiology And Clinical Experimental Research
> H-1094, Budapest
> Tűzoltó utca 37-47.
> Phone: [phone] ext.60342
> Mobile: [phone]

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