How long do you have the cells patched for?
I doubt that molecular weight is the limiting factor. Charge and the resulting tendency to interact with other cellular elements will also be part
of the problem. For labelling cerebellar granule cells (and visualizing their axons, parallel fibers) I used Atto 594 and Atto 488 quite successfully. Try not using hydrazide versions of the dyes, but rather carboxylic acids, as these interact less with proteins,
speeding up diffusion.
Lucifer yellow: small and old-school. It is fairly dim, so requires high concentrations. Which is why people use the lithium salt, as it is more
soluble than the potassium salt. Lithium interacts with calcium signalling (IMPase), so depending on what you are looking at, it might not be the best idea.
Just my thoughts and I am sure others will have additions.
Cheers, Christian
From: Peter Rupprecht [mailto:[hidden email]]
Sent: 21 December 2016 08:51
Subject: Highly diffusible dye for filling neurons in vivo
***** To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on
http://www.imgur.com and include the link in your posting. *****
Dear dye experts,
I'm currently patching very small neurons (soma 5-10 um in diameter, dendrites much smaller) in living tissue. In addition to electrophysiological
recordings, I'd like to get the morphology without fixing the tissue, so I add a dye (until recently mostly Alexa 594) to the pipette solution.
However, since the neuronal processes are very small, it is very often only the soma and not the small dendrites that get filled by the
dye.
Switching from Alexa 594 to Alexa 488 improved things a bit (probably since 488 is smaller? https://en.wikipedia.org/wiki/Alexa_Fluor).
But maybe somebody on the list has an idea of an even smaller, highly diffusible, non-toxic dye that I could use for this purpose. Plus, it should of course work with regular 2P excitation (800-950 nm).
Any ideas?
Best,
Peter
| Free forum by Nabble | Edit this page |