Application of Viral Vectors to Motor Neuron Disorders - Viral Vector Approaches in Neurobiology and Brain Diseases - page 227

l virus on a piece of parafi lm.
Gently suck up the virus with the insulin syringe making sure
no air is aspired.
2. Animal handling and vector injection (see Note 18 ) . Pups
should be put asleep as described above. Fix the pup to be
injected on a hard mount by applying tape on its back (Fig. 2 ).
The pup should be lying fl at on the surface with all four limbs
stretched out. Locate the point of injection by applying some
pressure with a marker at one-third of a fi ctive line that extends
from the confl uence of sinuses to the right eye. The skull
should be easily pushed in by slight pressure if correctly located
on top of the lateral ventricle. Mark the point of depression
with the marker. The pup should be facing the experimenter
for injection. The needle should enter the skull at the deter-
mined mark and penetrate 2 mm ( see Note 19 ). Make sure the
syringe is perfectly perpendicular to the skull. Inject the virus
as a bolus and slowly withdraw the syringe ( see Note 20 ).
Check the injection by applying the pup's head close to a light
source ( see Note 21 ). Reversal of anesthesia is performed as
described for intramuscular injections.

Green FCF. Pipette up to 4 μ

1. Perfusion . Mice are euthanized by intraperitoneal injection of
pentobarbital (150 mg/kg). Perfuse the animal with PBS sup-
plemented with 5UI/ml heparin until the extremities become
white; then continue the perfusion with a solution of 4 % PFA.
Stop the perfusion when the animal becomes rigid (around
30 ml of 4 % PFA per animal).

3.3.3 Immunohis tological
Detection of Retrograde
Transport Effi ciency

2. Spinal cord recovery . After decapitation, successively cut both
pedicles of each vertebra using small sharp scissors. Progressively
lift the detached transverse and spinous processes with a for-
ceps. The spinal cord appears lying on the vertebral bodies.
Detach the spinal cord by gently sliding forceps between the
spinal cord and the vertebral bodies.
3. Spinal cord handling for immunostaining . Postfi x the recov-
ered spinal cord in 4 % PFA for 1 h at room temperature.
Further transfer the tissue in a 30 % sucrose solution to pre-
vent freezing artifacts. Incubate overnight at 4 °C for the tis-
sue to absorb the sucrose. Mount the spinal cord in Cryomatrix
and freeze on dry ice. The spinal cord is cut free-fl oating in
25 m-thick sections, which are stored at 4 °C in a PBS-azide

solution for further histological processing. Regular immuno-
histological protocols can now be applied to detect expression
of the transgene.

μ