I'm trying to load some Red Dyes onto Rat brain after craniotomy. Dyes were spread over dried dura mater or sub-dura mater. But I cannot get any meaningful images from those trials. Because I had an experience about cardiac fluorescence imaging with same system, same dye (di-4-anepps) and It makes Meaningful Images, so I think procedure for dye-loading onto brain is problem.
Could you suggest the procedure or something that I should notice ?
The dye is hydrophobic so you should apply it in a solution of DMSO. Give the sample some time to soak (the time depends on the way you prep your tissue - Live cells may be incubated for short times (20 min) while retrograde loading in neurons will be longer (up to 24 hrs)). Then you should wash it with an aqueous solution with a lower (say 1/4) concentration of DMSO and no dye to remove the excess dye from areas that are more hydrophilic (you may want to wash again without DMSO). You should end up with a higher concentration of dye in the lipid layers and a lower concentration in the extracellular matrix and the cytosol. You should be careful not to use too high of a concentration of DMSO since it may permanently disrupt membranes.
Thanks for your kind answer.
i'm using EtOH instead of DMSO and it works well in cardiac fluorescence imaging.
Could you teach me why DMSO is better thant EtOH?
And also, I'm trying to image in vivo, so I load dye topically (in dorsal part of brain) so I think I cannot use retrograde loading
In my experience, DMSO has a higher mobility in the lipid bilayer and acts as a better carrier agent than alcohols. Generally DMSO solubilizes most hydrophobic compounds better than ethanol and can therfore be used at a lower concentration.
Ethanol also disrupts some ion channels (Crews et al., 1996, Podzorova et al., 2000, Woodward et al., 2008) as well as interfering with some receptor binding sites (Lovinger, 1997 Tabakoff et al., 1991 and Faingold et al., 1998).
Your issues could also be that since the brain has a much higher lipid content than cardiac tissue, your dye distribution is not as you're expecting. Maybe you'll need a different concentration. Also, since the solvent allows the dye to go everywhere, there is little specificity in what is getting stained. What do your images look like? No stain or too much, or just no definition of structure?
I have seen some in vivo DiI protocols where they apply the solid crystals of the dye to the brain surface to stain neurons that are connected to the point at which the crystals are placed (from my wife's lab, i cannot provide any references for this). Lateral diffusion in the cell membrane takes at least 6 hours and may require up to 24. Maybe this method could be better than applying the dye in a solvent solution.
Thanks again with your great answer!
It looks like loaded well, (big background illumination) but it doesn't react to any kind of stimulus that I gave. so I guess dye was bind onto another membrane, not neuron cell.
Anyway, I will find more things that you provide the clue for me.
Thanks!
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