AMPK Methods and Protocols

an autosampler (AS-2057 plus) from Jasco (Groß-Umstadt,

Germany), and a C 18 Nucleosil 100-3 (125/4) column from

Macherey and Nagel (Du ̈ren, Germany).

2.3 Dihydroethidium
(DHE) Staining
for the Detection
of Superoxide Anions
in Vascular
Cryosections

1. Krebs-Hepes buffer (KH).


2. Aprotinin: 1 mg/mL H 2 O (stock solution).


3. Pepstatin: 2 mg/mL EtOH (stock solution).


4. Leupeptin 5 mg/mL H 2 O (stock solution).


5. Krebs-Hepes inhibitory buffer (KH-I): KH, 10μg/mL apro-
   tinin, 8μg/mL pepstatin, 5μg/mL leupeptin. Take 100μLof
   aprotinin, 40μL of pepstatin, and 10μL of leupeptin, and fill
   up to 10 mL with KH buffer (seeNote 2).


6. Dihydroethidium (DHE): 1μM DHE in PBS. Since DHE is
   UV sensitive, it is essential to use dark 1.5 mL tube cups. For
   preparation we suggest to start with a stock solution of 5 mM
   DHE in DMSO. To get a final concentration of 1μM DHE in
   PBS, different dilution steps are necessary, e.g.:
   
   
   • Dilute 20μL stock solution (5 mM) with 80μL of DMSO
     to get a 1 mM solution (1:5).
   
   
   • Dilute 20μL of the 1 mM solution with 80μL of DMSO
     and 100μL of PBS to get a 100μM solution (1:10).
   
   
   • Dilute 20μL of the 100μM solution with 180μL of PBS to
     get a 10μM solution (1:10).
   
   
   • To get the final concentration of 1μM, dilute 100μL of the
     10 μM solution with 900μL of PBS. The 1μM solution is
     the working solution which is used on the aortic cryosec-
     tions (seeNote 3).
   
   
   
   

2.4 Assessment
of Protein Nitration via
Dot Blot Technique

1. SDS solution: 10% sodium dodecyl sulfate (SDS) in PBS.


2. Wash buffer (PBS-T): PBS, 0.1% (v/v) Tween-20.


3. Blocking buffer: (PBSþ5% milk): PBS, 5% milk powder.


4. Antibody: anti-nitrotyrosine antibody (e.g., millipore 05-233).
   Antibody solution should be freshly prepared at 1μg/ml in
   PBSþ5% milk (seeNote 4).


5. Contents to determine protein concentrations for standard
   Bradford assay or Lowry assay.


6. S-Monovettes for plasma preparation.


7. Homogenization buffer (Hg buffer): 20 mM Tris-HCl,
   250 mM sucrose, 3 mM EGTA, 20 mM EDTA.


8. Homogenization solution (Hg solution): 0.5 mM PMSF,
   2.5 mM sodium fluoride, 0.5 mM sodium vanadate, 1% (v/v)
   Triton-X in Hg buffer (see above). Add appropriate amount of
   phosphatase inhibitory cocktail and protease inhibitory
   cocktail.

498 Swenja Kro ̈ller-Scho ̈n et al.