In fact, I am have a good struggling with coinjection of fluorogold &BDA in the same brain region. My protocol for this combination is quite similar to which L. Swanson reported in PNAS, 2010 (http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2930585/), but with smaller injection current (+1-2 µA) and longer injection time (15-20 min). I can, however, get barely BDA labelled neurons or dendrites, but always very nice retrograde FG labelled cell bodies.
I almost always oberseve FG gets precipitated, especially up to first 5mm of glass pipette from its tip. I consider this might be a reason for a lack of BDA labeling in my case.
Any suggestions or comments? Thanks in advance!
Dear Jiahao,
I have always pressure injected Fluoro-gold, and have found that as long as it is solubilized in 0.9% saline it will not precipitate. I am not sure why yours precipitates with iontophoresis (perhaps big ionic changes with time?), but I could see where that could prevent the BDA from exiting the pipette. Fluorogold is such an efficient retrograde label that you are probably getting enough into the neuron in the first few minutes of application that you can find it in the cell body.
If there is some reason that you can't use +5 uA current for the Hz and time in the PNAS paper, I would trouble-shot this by just using the BDA alone with your parameters to see if you are actually injecting enough BDA into the neurons to get detectable labeling. The reason I suggest this is because it took us a long time to work out the parameters for filling goldfish retinal ganglion cells and tectal neurons with fluorescently conjugated dextran amines, and we had to try different Voltages/amperages and injection times/Hz. Of all things, we found that, along with optimizing our iontophoresis parameters, we had to use thin wall glass microelectrodes (with a filament for filling). I don't quite understand the physics of it, but it did make a significant difference.
Good luck!
Regards,
Jill
Dear Jill,
Thank you very much for your analysis and suggestions of troubleshooting.
Yes, I have good BDA labeling of cell bodies and dendrites when I inject BDA alone, with even smaller pipette openings of 5-10µm (And I have to work with glass pipettes of small openings to get a focal injection).
So I suspect the cloging of somehow preventing BDA into the brain. And I will try pressure injection as well. Fluorogold in solution is quite acid (pH 4.6 for 1% FG in water, Fluorochrome), do not know if this might impede the delivery of BDA into cell in coinjection.
BTW, thanks for the tips of thin wall glass pipette, I will keep it in mind as we only have a pile of capillary glass with standard wall+ filament.
Best,
Jiahao
I agree with Jill! I am only aware of the pressure injection of Fluorogold. Iontophoresis might indeed cause precipitates and clog your pipette tip. If you really want to co-inject, BDA and a retrograde tracer, I would advise you to switch to FastBlue. Hope this was helpful!
I generally have had less precipitation during iontophoresis with Fluorogold dissolved in 0.1M cacodylic acid, as compared with saline (just be sure never to pressure inject this particular solution). I don't know how well this would work for BDA however.
If you have a sonicator, this would likely help just prior to filling the pipette.
Also, if the tip size does not necessarily need to be the same for every animal, you could just pull the pipette back out and snip the tip with forceps whenever the resistance starts to increase... although this increases the physical damage with each snip.
Best of luck!
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