Check out "Novikova L et al. Persistent neuronal labeling by retrograde fluorescent tracers: a comparison between Fast Blue, Fluoro-Gold and various dextran conjugate. J Neurosci Methods 1997 74(1): 9-15." They don't specifically use Alexa 488 conjugated to dextran, but they do use three other dextran conjugates that should give you a good idea about the stability of the tracer over time.

Using Alexa dextran retrograde labeling in nerve fibres of tadpoles, our experimental results show that highest staining intensities are reached after 1 day and that the staining starts to decrease after 10 days but stays visible for another 5-10 days.

The best strategy in my opinion will be to just do it in a couple of subjects. As far as I know there is an antibody against Alexa488, which can be used to overcome possible decay of fluorescence. One month is a relatively short time for an anatomical project.

Cheers Sebastian! By decrease, do you mean that staining spreads non-specifically to other areas, or that the staining decreases in the area of interest?

You are quite right Victor, a preliminary study with a couple of subjects would answer the question emphatically. My main concern is the dextran spreading over time such that my labelling is no longer labelling what I think it is, but a preliminary study should address this. I was not aware of there being an anti-Alexa488 antibody, but I've just looked it up and found it to be true, which could be an added bonus.

By decrease I mean decrease in an area of interest, e.g. an axonal fiber tract.

for the immunoprotocol on DRGs I prefer frozen ~15-20um

Thanks for the responses. That thickness of slices is what I've done before, thanks for your tip.

you can also try injecting CTB (cholera toxin beta subunit) conjugated with fluorophore like FITC (488)

Hi Dvir, is there are particular reason you would recommend CTB conjugates ahead of dextrans?

basically, CTB will be taken by the synapses using endocytosis process.i do not know whether you will get tha same amount of labeling after a month since i did not go that far but i can say that the labeling 10 days post injection is very good and stable. i think it worth the try if you are looking into other options.

Okay, thanks for the info.!

Check out "Novikova L et al. Persistent neuronal labeling by retrograde fluorescent tracers: a comparison between Fast Blue, Fluoro-Gold and various dextran conjugate. J Neurosci Methods 1997 74(1): 9-15." They don't specifically use Alexa 488 conjugated to dextran, but they do use three other dextran conjugates that should give you a good idea about the stability of the tracer over time.

Thanks a lot Zachary, I'll grab that paper now!

Yes Fluoro-Gold is a very good and stable marker, in my experience it remain stable within the cells for 6/7 months when the animals are still alive. One bad thing about this marker is some time it diffuse to neighboring area to avoid that you may try concentrated solution like 10%

Cheers Farid, much obliged for the tip!

From what I understand, retrograde labeling with a dextran such as Alexa488 or BDA requires axonal damage for the tracer to be taken up. I have used it a lot in an acidic medium as it increases this uptake and results in great cell morphology. See: "Improved retrograde axonal transport and subsequent visualization of tetramethylrhodamine (TMR) -dextran amine by means of an acidic injection vehicle and antibodies against TMR" J Neuro Methods 1996; 65(2) pp 157-165. I have only used this in short term experiments though. If you are hoping for a long term label I would use CTB or fluorescent microspheres. I have gotten great results with the microspheres with the main drawback being that they don't fill the cell, so are not useful for dendritic morphology. Check out: "Fluorescent latex microspheres as a retrograde tracer in the peripheral nervous system" Brain Res. 486(2) pp 334-339. Hope this helps.

Thanks for the answer James. When I've done dextra-Alexa injections before I've never added any intended damage, other than that resulting from the mechanics of the injection itself - labelling in dorsal root ganglion neurones was great 3 days post injection, I'm just wondering how long the labelling stays so robust...

Hi Ewan,

I have used Alexa Fluor coupled to Dextran (10,000 MW but I believe 3,00 MW would behave the same) for retrograde motor neurone staining from Xenopus tadpole dorsal muscles and made relevant observations in young post-metamorphosis juveniles, so between 1 and 2 months later. I don't know what you'll be able to see in mammalian cultures but in amphibians, fluorescence is still clearly detectable 2 months after injection.

Thanks a lot Didier, I think in the long run that I will just have to give it a trial run and see what happens, but what you write is encouraging - thanks a lot!

Ewan,

years ago, before I got involved with MP to develop the 3,000 dextran amines, we used 10,000 dextran amines for long term labeling to investigate the regeneration of trochlear motoneurons in tadpoles. The dextrans survive long time but get aggregated into larger fluorescent lumps.

Here is the paper we published soem 20 years ago.

Sequential double labelling with different fluorescent dyes coupled to dextran amines as a tool to estimate the accuracy of tracer application and of regeneration.

Fritzsch B, Sonntag R.

J Neurosci Methods. 1991 Aug;39(1):9-17

Hi Bernd, thanks very much for the info.! For what I wish to do, do you think that there would be any major difference between using 3,000 and 10,00 dextran amines?

Ewan,

based on limited sample size, my impression is that for whatever reason 10,000 dextran amines seem to last somewhat longer, at least in amphibians. How this compares to mammals is unclear as I have only done short time in mammalian embryos, banking on the faster diffusion with the lower molecular weight dextran amines. I have not seen any direct comparison of dextran viability over long term in mammals, this would be up to you.

Cheers for the honest advice Bernd, much appreciated!

Honesty is my middle name :-)

Best of luck with your research, Ewan.

Hi Ewan,

In my experience -limited to tracing within the CNS- retrograde tracing with biotinylated dextran amine is stable with a survival time of up to 8 weeks. Longer survival times were never tested by us. We routinely use both 3 KDa and 10 KDa BDAs from Molecular Probes-Invitrogen. In our experience, our favourite choice is to use biotinylated dextran amines followed by detection with alexa conjugates of streptavidin.

Best, Jose L.

Cheers José - much appreciated!

stored cool and dark your staining can last up to half a year. So no worries when it is just 1 month.

I just wanted to provide a bit of an update here, which is that using Alexa-488 dextran caused a problem for visualisation of fixed (4% PFA) cells, such that there was significant autofluorescence - even neurones from a non-injected mouse showed a lovely glow after dissociation and fixing!

We've now trialed Red retrobeads from https://lumafluor.com/Information.php and initial results are encouraging, e.g. no autofluorescence, but even if there was some, the presence of the beads within cells would make it easy to distinguish autofluorescence from beads. The next steps are to examine how long post-injection we can see staining and then to patch these bead-filled neurones; other publications suggest that this will not be a problem....

Hi Ewan, What worked best for you finally? I am trying similar thing -- injection in the foot pad and isolating DRGs.....

 Hi Ewan St. John Smith, could you tell me what worked best for you finally? I am trying  Dextran tetramethylrhodamine and biotin 3,000 MW from Life Technologies for tracing neurons in DRGs after foot pad injection

I've ended up switching to Lumafluor beads: https://lumafluor.com/Information.php - we get robust labelling after about 3 days and looks fine up to 3 weeks, not looked any further. Good luck.

Hi,

when I worked out with Molecular Probes the production of 3k dextran amines (Fritzsch, 1993) we had already established that dextran amines are excellently suited for regeneration experiments.  Here is a early reference that gives you on overview of different techniques using fluorophores. (Fritzsch, Sonntag, J Neurosci Method 39:9-17).