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Hi Maria,
For staining 200 um thick brain slices I use usually Alexa- 488 and/or
594 and mount them in PromoFluor Antifade Reagent from Promokine
(
http://www.promokine.info/products/fluorescent-labeling/promofluor-antifade-reagent/).
On our Leica SP5/Coherent Chameleon Ultra II system (Ti:sapphire laser)
I use 770 nm for excitation and NDD1/2 for separation. So far I never
had problems with bleaching, weak signal or cross talk after 48h of
mounting.
I always check these links to find the right "colour-combination" or
wavelength for MP imaging.
http://www.drbio.cornell.edu/cross_sections.htmlhttp://www.spectra.arizona.edu/:) Manja
On 16/08/2012 00:01, Ingaramo, Maria (NIH/NIBIB) [F] wrote:
> *****
> To join, leave or search the confocal microscopy listserv, go to:
>
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy> *****
>
> Hello everyone!
>
> I have two questions.
>
> I am trying to image microtubules in fixed cells using tunable two photon excitation. I am interested in optimizing signal, and was wondering if anyone had suggestions regarding really good two photon excitable dyes for immuno-staining them. Alternatively, I could transfect with a fluorescent protein fusion.
>
> Also, I have tried imaging Alexa-488 and EGFP labeled tubulin in fluoromount G, and I am not happy with the results obtained in this media. Does anyone have a favorite mounting media that yields good images upon two photon excitation?
>
> Thank you!
>
> -maria
--
Dr. Manja Schubert
University of Bergen
Department of Clinical Medicine
Haukeland University Hospital
Section Neurology - Paraneoplastic Syndrome group
5021 Bergen
Norway
Tel:+47-55 58 67 15
Fax: + 47-55 58 63 60
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