Fluoro-Ruby 30 mg
A 10% solution of Fluoro-Ruby is made by dissolving 10 mg of Fluoro-Ruby dry powder in 100 µL of distilled water. It is then delivered via intracranial stereotaxic injection using a 1 uL Hamilton microsyringe to pressure inject the tracer. Injection volumes typically range from 0.02-0.1 µL and are gradually injected over a 10-15 minute interval. The animal is allowed to recover and is sacrificed typically 4-14 days later.
The animal is then perfused with neutral buffered formaldehyde (10% formalin or 4% paraformaldehyde in 0.1 M neutral phosphate buffer.) The brain is removed and postfixed for at least overnight in the same fixative solution plus 20% sucrose for cryo-protection. The brain is then cut on a freezing sliding microtome or cryostat into sections usually between 20 and 40 microns in thickness.
The tissue sections are mounted on gelatin coated slides and are air dried on a slide warmer at 50-60 degrees Centigrade for at least one half hour. The dry slides are then transferred to xylene clearing solution for at least one minute and then coverslipped with DPX mounting media. The slides can then be examined under an epifluorescent microscope using a filter suitable for visualizing TRITC (green light excitation).