3 Methods
Carry out all procedures at room temperature unless otherwise
specified.3.1 Vascular Nitric
Oxide Analysis by EPR
Spectroscopy with Fe
(II)(DETC) 2
as Spin Trap
For good results you should perform every step very clean and
quickly. Be prepared with everything you will need for the follow-
ing steps.3.1.1 Sample
Preparation
- Remove all connective tissue as well as vascular fat tissue from
aortic sections. Dissect the aorta into pieces of 5 mm. - Put 1 mL of KH in each well of a 12-well plate, and add two
aortic sections of 5 mm into every well (two pieces per well). - Add 2μL of the Ca ionophore to the solution and indirectly to
the aortic sections (seeNote 5). - Add the prepared 15 mL of FeSO 4 solution quickly to the
15 mL of DETC solution, and mix it carefully. Avoid too
much bubbling (seeNote 6). - Give 1 mL of the FeSO 4 /DETC solution to each well.
- Incubate the samples for 1 h at 37C.
- After incubation of the samples, the aortic rings are embedded
in KHinside of the capless syringes: put 200μL of KH into the
syringe; withdraw it with air to 700μL. Avoid air bubble inside
of the solution. Freeze it upside down in liquid nitrogen. - Clean the syringe with a wipe from condensed water.
- Add 100μL of KH without air bubbles, and put two pieces of
the aortic sections to this solution using forceps, and freeze the
syringe again upside down in liquid nitrogen. This makes sure
that the solutions are fixed in the syringe. - Clean the syringe with a wipe from condensed water.
- Add again 160μL of KH to the syringe.
- Freeze the syringe again upside down in liquid nitrogen.
3.1.2 Measurement 1. Apply the following parameters: B 0 -field: 3274 G,
sweep¼110 G, sweep time¼60 s, modulation¼7000 mG,
power¼10 mW.
- Cool down the EPR tube with liquid nitrogen.
- Remove sample carefully out of the capless syringe by pushing
the plunger without breakage, and transfer it to the EPR tube. - Fill up the EPR tube with liquid nitrogen, and cover everything
with cotton batting.
NO & Oxidative Stress 499