Photoreceptor Degeneration in Mice: Adeno-Associated Viral Vector-Mediated Delivery of Erythropoietin - Tissue-Protective Cytokines: Methods and Protocols - page 237

Incubate the membrane in the secondary antibody diluted
1:3,000 in 5% milk/TBS-T at room temperature for 45 min
on a shaker.
17. Wash the membrane three times in TBS-T (10 min each time)
at room temperature on a shaker.
18. Incubate the membrane in ECL for 5 min at room
temperature.
19. Visualize the protein bands by juxtaposing the filter to X-ray
films and by developing X-ray films under red dim light follow-
ing standard procedures (see Note 12).
20. Once the expression of the EPO and S100E proteins has been
confirmed generate AAV vectors using either the pAAV2 . 1 -
CMV - EPO or the pAAV2 . 1 - CMV - S100E (see Notes 13-15).

1. Thaw the required number of AAV vector aliquots; the AAV
vectors required are the AAV-EPO, the AAV-S100E and the
AAV-EGFP as negative control (see Notes 4-6).
2. If necessary, dilute AAV vectors using 1× PBS as follows (steps
3 and 4).
3. For the subretinal injections dilute each AAV vector to 1 × 10 9
GC (genome copies)/

3.3 Subretinal
and Intramuscular
Injection of AAV
Vectors in Albino Rats

3.3.1 AAV Vector
Preparation

L will be injected
in each eye (thus, the dose will be 2 × 10 9 GC/eye).
4. For the intramuscular injections dilute:
(a) AAV-EPO to 1 × 10 8 GC/

μ L considering that 2 μ

L per
rat are required (thus, the dose will be 1 × 10 10 GC/rat)
(see Note 16).
(b) AAV-S100E to 1 × 10 9 GC/

μ L, considering that 100 μ

L per
rat are required (thus, the dose will be 1 × 10 11 GC/rat).
(c) The control vector AAV-EGFP to 1 × 10 9 GC/

μ L considering that 100 μ

μ L consid-

L per rat are required (thus, the dose will
be 1 × 10 11 GC/rat).
5. Put the working dilution on ice, store the leftover of the AAV
vector stocks at −80°C.

ering that 100 μ

1. Anesthetize the 4 week-old rat with an intraperitoneal injec-
tion of 2 mL/g of body weight of Avertin (prewarmed at
37°C). Wait until the rat is properly anesthetized.
2. Place the anesthetized rat under a dissecting microscope and
position the light fibers as preferred. Make sure that the eye to
be injected is clearly observable.
3. Delicately grab the temporal conjunctiva using tweezers.
4. Using scissors carefully perform a single cut of the conjunctiva
at the level of the pars plana (Fig. 3 ).

3.3.2 Subretinal
Injections
( See Notes 17 - 20 )