[Histonet] Rat brain vascular casting using Mercox

[Chris Bjornsson]

Hi Histonetters,

I've been trying to get complete/satisfactory Mercox casts of rat cerebral
cortex, and have spent considerable amounts of time and reagents
troubleshooting with limited success. A difficult technique! We want to be
able to compare results among different animals and have therefore been
using a constant-pressure perfusion system. I've been performing
transcardiac perfusions with 37degC heparinized buffer, followed by
room-temp 4% PF fixation so we can stain different cell structures, and
using rhodamine B-labeled Mercox diluted 20% with MMA. I've tried changing
pressures, fixed vs. unfixed tissue, clamping the descending aorta, etc. to
no avail. So far it appears that Mercox pressure is actually less critical
than the flow rate of buffer & fix, and that clamping the descending aorta
improves vessel filling but doesn't completely solve our problems. We've
also noticed that little things like perfusion needle angle and placement
have a profound impact on flow rate and cast quality. I haven't explored
temperature effects much as of yet. Does anyone have any experience or
advice on this topic?

Thanks, cheers,
Chris Bjornsson
Laboratory of Nervous System Disorders
Wadsworth Center
Albany, NY 12201