Photoreceptor Degeneration in Mice: Adeno-Associated Viral Vector-Mediated Delivery of Erythropoietin - Tissue-Protective Cytokines: Methods and Protocols - page 231

2.2.2 Protein Extraction,
Quanti fi cation, and
Western Blot Analysis

1. RIPA buffer: 50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1%
NP40, 0.5% Na-Deoxycholate, 1 mM EDTA pH 8.0, 0.1%
SDS dissolved in H 2 O.
2. Protease inhibitors (e. g. Complete Protease inhibitor cocktail
tablets, Roche; dissolve 1 tablet in 500 μ L of H 2 O to achieve a

100× stock solution, store at −20°C).
3. 200 mM (200×) Phenylmethanesulfonylfluoride (PMSF) dis-
solved in isopropyl alcohol (store the solution at −20°C).
4. Standard equipment and reagents for protein quantification: a
colorimetric assay (e.g., BCA protein assay reagent, Thermo
fisher scientific, USA or BIO-RAD protein assay, Bio-Rad,
USA); a spectrophotometer or a microplate reader (supplied
with 560 and 595 nm filters).
5. Standard equipment and reagents for Western Blot analysis
(SDS-PAGE and immunoblot).
6. 1× Tris-HCl Buffered Saline-Tween (1× TBS-T): 50 mM
Tris-HCl, 150 mM NaCl, 0.05% Tween 20.
7. 5% nonfat dry milk dissolved in 1× TBS-T.
8. Anti EPO primary antibody (EPO N-19, sc-1310 Santa Cruz
Biotechnology Inc., USA).
9. Secondary antibody (e.g., Anti-Goat IgG HRP Affinity
Purified, HAF109, R&D Systems, USA).
10. Enhanced chemiluminescence (ECL) substrate.

1. Purified AAV vectors encoding EPO, S100E, or EGFP as neg-
ative control (see Notes 4-6).
2. Sterile 1× phosphate buffered saline pH 7.4 (1× PBS): 137 mM
NaCl, 2.7 mM KCl, 4.3 mM Na 2 HPO 4 , 1.47 mM KH 2 PO 4
dissolved in H 2 O.
3. H 2 O.
4. 70% Ethanol.
5. Avertin (Anesthetic): 1.25% w/v of 2,2,2-tribromoethanol
and 2.5% v/v of 2-methyl-2-butanol. To prepare a working
solution mix 2.5 g of 2,2,2-tribromoethanol in 5 mL of
2-methyl-2-butanol in the dark (i.e., use a bottle wrapped in
foil), let the powder dissolve heating it at 50°C on a magnetic
stirrer. Once the powder is completely dissolved add 200 mL
of an isotonic saline solution (0.9% NaCl in H 2 O) and let the
solution mix overnight (about 12 h) at room temperature
using a magnetic stirrer. Filter the solution through a 0.22

2.3 Subretinal
and Intramuscular
AAV Injections

2.3.1 Intramuscular
and Subretinal Vector
Administration

μ m

filter. Store at 4°C wrapped in foil.