Application of Viral Vectors to Motor Neuron Disorders - Viral Vector Approaches in Neurobiology and Brain Diseases - page 228

Fig. 3 Transgene expression in the mouse spinal cord following intracerebroventricular (ICV) or intramuscular
injection. Representative spinal cord sections immunostained with an anti-GFP antibody following ICV
( upper left and right panels ) or intramuscular ( lower left panel ) injections. Note that ICV injection leads to GFP
expression in motor neurons as well as sensory fi bers at all levels of the spinal cord. Scale bar: 300 μ m

4. GFP immunostaining . Rinse spinal cord sections three times
in PBS before incubating them for 1 h at room temperature
and on gentle shaking in a blocking solution containing 3 %
normal goat serum (NGS) and 5 % BSA in PBS. Directly trans-
fer sections in the primary mouse anti-GFP antibody diluted
1/500 in PBS/3 % NGS/5 % BSA/0.15 % Triton ® X-100.
Incubate overnight at 4 °C with gentle shaking. Rinse spinal
cord sections three times in PBS before transferring them to
the secondary Alexa Fluor 488 goat anti-mouse antibody
diluted 1/500 in PBS/5 % BSA/0.15 % Triton ® X-100.
Incubate for 1 h at room temperature with gentle shaking.
Rinse three times in PBS before mounting sections on micro-
scope slides. Let sections dry before rinsing them in water and
mount them with Mowiol.
Examples of GFP expression following either intramuscu-
lar or ICV injections of an AAV6-cmv-GFP vector are shown
in Fig. 3 .