We're trying to inject the cascade blue dextran into mouse visual cortex by the protocol described in Wang & Burkhalter, 2007, but seem to have issues with the pipette clogging. We'd like to do it by iontophoretic injections so that we can closely control the injection amount, in order to only inject a small area. Any advice helps, thanks!
http://onlinelibrary.wiley.com/doi/10.1002/cne.21286/abstract
I suggest using a final dextran concentration of 1-5%. In our hands higher concentrations lead to pipette clogging. We used patch micropipettes to inject micro ruby and micro emerald iontophoretically, but have not tried this protocol with cascade blue yet.
Good luckAdrian
What is the molecular weight? I've used successfully 10000 and 3000 with several fluorescent tags. We use to dissolve it in 0.01M phosphate buffer, pH 7,6 at a concentration of 5%. We use a pipette of about 10 micron-thick or so. Then we inject with positive square pulses of current, 7s ON 7s OFF, 2-5 microamperes for 5-20 min, depending on the size of the injection site we want to obtain.
What are the conditions you're using?
Good luck
Fernando
Fernando, we are using MW 10,000. Initially I used a pipette of about 20 microns, perhaps that was too big - I'll have my answer later today. Those are about the conditions I have been using. I'm looking to infect about 100-200 um diameter of cortex.
I did a few injections with low concentration (4-5%) in H20 and it seemed to work well without clogging, so thanks for that tip, Adrian!
Hi Ashley
I think it would be wiser and more reproducible by using a low-molarity buffered solution, e.g. PB 0.01M. The reason is that at a fixed pH the charge of the molecule is controlled, thus ionophoresis is more reliable.
No problem with a 20 micron-thick pipette. But the thinner the better for a small injection. However, when the tip is very thin resistance is high and you need a more powerful current generator.
Good luck,
Fernando
Good to see this post, I had trouble with the size of the tip: at 20um, the dye will leak out without applying current. No idea how to control it...
I am trying to inject MW 10,000 dextran into the orbitofrontal cortex. I am using a 13 microns glass pipette. I am injecting just with a regular syringe and a tubing which is connected to the pipette. I fixed the brain in PFA, cut in the freezing microtome and I don't see any fluorescence. What could be the problem? Old dextran, need antibodies to amplify the signal or I have to image slides in a special way. Note: I am doing this for the first time! Thank you for your answers.
Hi Irina
What concentration of dextranamine are you using? Are you using a lysine fixable one? What tag has the dextran? A fluorophore? Biotin? What volumes do you (try to) inject? What injection system are you using?
These questions are important to help you
What concentration of dextranamine are you using? I am using dextran, Alexa Fluor 488, 10000MW, anionic, fixable.
What volumes do you (try to) inject? 0.5
What injection system are you using? Simple syringe, with tubing and a glass pipette
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