3.4.2 Preparation
of Tissue Samples
- Homogenate frozen tissue samples on liquid nitrogen using a
mortar and pestle. Tissue should be completely pulverized. - Transfer the samples to a 1.5 mL tube and dilute with Hg
solution. Volume of Hg solution should correlate with the
amount of powder. - Resuspend the tissue powder thoroughly.
- Centrifuge at 10,000gfor 10 min at 4C.
- Transfer the supernatant to a new 1.5 mL tube and discard the
cell debris. - Proteins are solved in the supernatant.
3.4.3 Assay 1. Determine the protein content of each plasma sample accord-
ing to standard Bradford or Lowry assay.
- Adjust the protein concentration of each sample to 0.5μg/μL
with PBS (prepare enough sample volume to have 50μg pro-
tein per dot, application of 100μL per dot). - Add the appropriate volume of 10% SDS to get a final concen-
tration of 0.01% SDS per sample. - Samples can be stored at 20 C or used immediately.
Fig. 3Visualization of vascular superoxide production by DHE staining. Dihydroethdium-stained aortic
cryosections from wildtype mice are shown on the left side. Aortas fromα1AMPK knockout mice displayed
increased oxidative stress in all layers of the vascular wall as indicated by the intense red fluorescence signal
NO & Oxidative Stress 503