Re: RE Ca-measurement in glandular tissue

Posted by Axel Kurt Preuss on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Ca-measurement-in-glandular-tissue-tp5035189p5035908.html

He is compartmentalising his fluo, so whether he sees fluorescence or notay be secondary
After 1.5 hrs loading time the poor fluo 4 molecules are probably  gasping in pain and can't take it anymore ( with it I mean the Ca) (scientifically actually they probably could "take" the Ca but without signal increase

Best to load cells the old fashioned  way per injection.

But If it has to be AM  best to try different concentrations
 in incubator for not more than 40 minutes
Yes, with so many cells in the tissue competing for fluo it could be fluo concentration is not sufficient. But he HAS to try different concentrations, as overloading could also be the case, and MUST avoid compartmentalisation

And I would stay away from PBS

It is aost  hopeless to try to "stain" tissue fragments with Ca dye (AM)
Bbbwouldb probably only work if there are cells extruding and not touching other tissue. Such exposed  cells might take up fluo the right way

Best to use a method to selectively target a few cells. This means injecting the free acid or experimenting with electrophoresis (dextran coupled dyes) or local perfusion with AM

Or to digest the tissue to the point of discrete cells

My text may be garbled due to compartmentilizing iPhone keyboard









Thanks

Axel

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

On May 11, 2010, at 8:38 PM, "[hidden email]<mailto:[hidden email]>" <[hidden email]<mailto:[hidden email]>> wrote:


Hi Attila,

Do you see any fluorescence? Does fluo3 etting inside the tissue?  Maybe adding some pluronic acid can help to have a better signal!

Have a nice day,

Louisssssssss


Cselenyak Attila <[hidden email]<mailto:[hidden email]>>@LISTS.UMN.EDU
Envoyé par : Confocal Microscopy List <[hidden email]<mailto:[hidden email]>>

2010-05-11 05:41

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        Ca-measurement in glandular tissue









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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