3.3.1 Sample
Preparation
- Remove all connective tissue as well as vascular fat tissue from
aortic sections. Dissect the aorta into pieces of 3 mm. - Prepare one 1.5 mL tube by adding 500μL of KH-I for each
sample. - Add one aortic section per tube and incubate for 10 min at
37 C. - Prepare aluminum foil cups for embedding the aortic section.
Be sure that the size of the so formed cups is small enough to fit
into the storage cups. - FillupthealuminumfoilcupswithTissue-TekO.C.T.compound.
- After incubation the aortic sections are embedded in a matrix
for cryostat sectioning. Therefore remove the aortic section out
of the incubation cups, remove the remaining buffer out of the
rings, and put them upright into the prepared Tissue-Tek
filled cups. - Freeze the cups carefully over liquid nitrogen (seeNote 8).
- If the Tissue-Tek O.C.T. is totally frozen, remove the alumi-
num foil and store the sample at 80 C. - For microscopical analysis and DHE staining, the prepared
aortic samples are cut in 8μm sections using a cryotome and
transferred to microscope slides. - Warm the microscope slide with your finger to fix the aortic
section.
3.3.2 DHE Staining The DHE dye is light sensitive. Working in a dark or at least shaded
room is preferred.
- Thaw the microscope slides and put 1 mL of the 1μM DHE
working solution on the slide and incubate for 30 min at 37C. - After 30-min incubation, remove the surplus solution and
cover the samples with a coverslip. - Take the pictures using a fluorescence microscope. Figure 3
shows representative DHE staining obtained from murine aor-
tic sections. When compared to the control group, global
AMPKα1 deficiency significantly increased red fluorescence as
an indicator of increased oxidative stress.
3.4 Assessment
of Protein Nitration via
Dot Blot Technique
3.4.1 Preparation
of Plasma Samples
- Prepare EDTA plasma samples using S-Monovettes according
to the manufacturer’s protocol, and determine the protein
concentration of each sample (seeNote 9).
502 Swenja Kro ̈ller-Scho ̈n et al.