Maria,

this is my area of expertise. What do you need to know that is not in my papers on this subject? Bernd Fritzsch

Hi Bernd,

Correct me if I am wrong but the protocols you have in your papers are for blocks of tissue. I am currently waiting for dyes to diffuse in a trial mouse brain following your protocol (J Comp Neurol 2004). I expect that at least I will be able to get images from that. Unfortunately at the point I joined the lab, pretty much all our "real" tissue has been sectioned already. By the way, Did I miss one of your papers dealing with sections?

I guess the dyes won't be able to diffuse as nicely in the sections as they would in the whole tissue but I expected the dyes still being able to label membranes, right? That's not happening. We are using the crystals directly on the sections but after mounting the dyes fade. At the begging I though it was the glycerol based mounting medium but we switched to a non-glycerol one and still happening. Is it because we are not embedding? Have you experience this sort of problem? Needless to say I am rather new in this, so any advice will be greatly appreciate it.

Thank you!

Maria,

if the tissue is already sectioned there are two ways using lipophilic dyes in my hands. First, you can take some of our preloaded filters and cut them into handy little pieces (see the video we made on this here: http://www.jove.com/video/2451/combining-lipophilic-dye-situ-hybridization-immunohistochemistry?ID=2451). Alternatively, you can dissolve dyes in detergent, rip out some hair, soak it in the dye solution, let it dry and poke a stub of hair right into the place were you think axons of the neurons you want to label are running. If you already have say 200um thick sections you can simply view them using buffer, but coverslip with spacers (I use other coverslips for this). Pressure will compress the section and destroy lipid bilayers and thus cause diffusion of dyes out of labelled profiles.

Let me know if that helps.

Bernd

Dear Maria

use Dil.

you can find more info at:

Chen, B. K.; Miller, S. M.; Mantilla, C. B.; Gross, L.; Yaszemski, M. J.; Windebank, A. J. Optimizing conditions and avoiding pitfalls for prolonged axonal tracing with carbocyanine dyes in fixed rat spinal cords. J. Neurosci. Methods 2006, 154, 256–263.

I am working with DiI tracing for many years. You can find in PubMed my papers Irina G.Makarenko. Embedding medium and cutting are very important! I prefere vibratome sections embedded in moviol.

I want to add, that according my experience frosen sections can not provide good DiI labeling because of significant lateral diffusion of the marker from neuronal membrane.

Hi Maria, I am very interesting with your question. Now, I am working with DiI staining for dendritic spines labelling. However, I tried many times and It was failed. I couldnt get a good staining of dendritic spines and the dyes diffuse in all region of interest (in this case, my target region is hippocampus) and spreading over a large area. Thus, I couldnt observe individual cells.

as for your information, I applied DiI using needle (our lab hasnt a gene gun or other specific tools for DiI staining) and before I apply, I always sonicate the powder. But still, I havent got a satified result.

If you have succeed this protocol, please let me know how to solve the problem, also for others who is expert in this field.

Looking forward for advices from all of you.

Regards,

Ria

Molecular Probes have a Chloromethyl version of DiL (cat. No V22888) which allows you to post-fix the tissue, with the dye firmly attached.

Hi everyone,

I have read that Dil may be diluted in DMF, DMSO or even ethanol. I saw that when dissolved with DMF it may require sonication.  Could somebody give me any advice for the dilution?

Thanks!!