in vivo brain stain question

Dear Histonetters,

I am a post-doc working in a rat model for stroke. Part of the animal model
entails running a small catheter up into the anterior cerebral artery (ACA)
right before killing the animal. For descriptive purposes, I would like to
inject a dye through the catheter, that will stain, not only the vessel
wall, but also will cross the blood-brain barrier and stain brain
parenchyma. Ultimately, I would like to photograph a coronal section of
brain showing dye stain of only the ACA distribution.

I have tried methylene blue and, when infused at rat mean arterial
pressure, it stains the wall of the ACA and some of its branches well, but
does not penetrate into brain parenchyma, presumably because endothelial
tight junctions are keeping it out. I tried raising the methylene blue
infusion pressure in the hope that I would rupture microvessels and get a
parenchymal stain, but my catheter is proximal enough that I just get
reflux into the rest of the circle of willis... not helpful. After
injecting the methylene blue, I did NOT try perfusing the animal with
paraformaldehyde (our normal procedure prior to post-fixation, processing,
and embedding).

So my three questions are: First, does anyone have any suggestions for a
dye I may use in vivo, minutes before death, that will penetrate brain
parenchyma? Second, in the event that such a dye is available, how should I
fix and process the tissue to retain the parenchymal stain? Third, in the
event that such a dye is not avilable, does anyone have suggestions on how
to process the methylene-blue stained tissue (stain limited to vasculature)
so as to retain the stain? Post-fix only perhaps?

To anyone who waded through this onerous message, thank you.

Walt Zink, PhD
University of Nebraska

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