Quick version: Looking for dye that can be infused in rat brain to check coordinates; that won't dissipate during formalin perfusion or soaking in sucrose to be cut with cryostat; and that won't spread all over the place.
Background:
I am going to be doing virus infusions in rat brains, but first, I would like a quick way to test my coordinates. I'm using male and female rats, and aiming to hit some brain regions that my lab does not frequently work with, including amygdala.
I have a Hamilton infusion pump and 28GA cannula that we lower into the brain with a stereotax for our AAV infusions, and I would like to use this setup to infuse some sort of dye - I can precisely infuse very small amounts (nanoliters) if that would be necessary to keep the dye local. I would like to be able to perfuse the animal with formalin or PFA as soon after this infusion as possible (I don't want to wait 4+ weeks like I would have to with a virus test to get transfection), and I would then cryoprotect the brain with a 30% sucrose formalin or phosphate buffer solution and use a cryostat for sectioning at ~40um.
I would then like to visualize the slices using a non-fluorescent scope, but I do have a fluorescent scope that works with flourophores in the green and red emission range (like Alexa Fluor 488 and 594). Ideally, I would see a small dot of dye where the tip of my cannula was, so I don't want the dye flowing all around.
Molecular Probes has a couple of DiI/DiO derivatives (CM-DiI and CM-DiO, I think) that are supposed to be stable over the long term and, I think are supposed to be "fixable" with paraformaldehyde.
If you try this, please post how it works. This is something my lab could find very useful.
It is difficult to work with dyes becaus most of them are spreading around the tissue. I recommand - if it is possible - to create small micro coagulations in the region of interest. If this is not possible take bigger cunulas to see the tract in the tissue and hopefully the tip as well. Take every 5th or 10th section for a Nissl-stain (Cresylviolett, Thionin, Toluidin blue, Methylenblue for bright field or NeuroTrace (Moleculare Probes/Invetrogen) for fluorescence). In this stained sectoins it is easy to find the tracts and to compare the regions with the coordinates in the Paxinos atlas. Good luck!
Thank you for your replies.
David, I haven't seen the CM-DiI/DiO used in vivo. Hoping for a cheaper way, or at least one where there's a more established protocol.
Ute, this is one method we've used, and it isn't as precise as we'd like. Excessive damage from a large cannula, not enough from small ones, tissue damage during perfusion or processing, and being too close to a ventricle have all made it difficult to visually exactly where the cannula tip was. It certainly is something to work with, I was hoping there was a commonly used dye-workaround!
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