- Expose to film or analyze with a chemiluminescent detection
system. - For quantification purposes, a densitometric analysis of each
dot is preferred. Figure4 shows a dot blot of control (negative
control) and nitrosylated EDTA plasma samples (positive
control).
4 Notes
- Krebs-Hepes buffer can be prepared 1 day before usage, but
adjustment of pH should be done right before usage. - Krebs-Hepes inhibitory buffer should be prepared freshly
every day. - Since dihydroethidium is a fluorescent dye dissolved in pure
DMSO, the dilutions have to be prepared freshly before use. - Antibodies can be reused. After first usage, add sodium azide
(6% solution) at a final concentration of 0.03% and store it at
4 C. - Endothelium-denuded aortic sections can be used as a negative
control. - Repeater pipette (e.g. Eppendorf®) is useful to allow a quick
addition of the Fe(II)(DETC) 2 for several samples. - If the aortic segments are shorter than 3 mm in length, a total
of three pieces per sample may be used. Since radicals are highly
reactive species and proteins are still biologically active, prepa-
ration steps and sample handling should be done quickly. - If the freezing process is too fast, the matrix for cryosectioning
(e.g., Tissue-Tek O.C.T.) will break and the sample is lost. - Centrifuge S-Monovettes for 10 min/1600 g at room
temperature. - Make sure that the sample volumes are flown through before
moving to the next step. - Do not puncture the membrane while applying the sample to
the grid.
References
- Kleschyov AL, Mollnau H, Oelze M,
Meinertz T, Huang Y, Harrison DG, Munzel T
(2000) Spin trapping of vascular nitric oxide
using colloid Fe(II)- diethyldithiocarbamate.
Biochem Biophys Res Commun 275:672–677 - Kissner R, Nauser T, Bugnon P, Lye PG, Kop-
penol WH (1997) Formation and properties of
peroxynitrite as studied by laser flash photolysis,
high-pressure stopped-flow technique, and pulse
radiolysis. Chem Res Toxicol 10:1285–1292- Beckman JS, Koppenol WH (1996) Nitric
oxide, superoxide, and peroxynitrite: the good,
the bad, and ugly. Am J Physiol 271:
C1424–C1437
NO & Oxidative Stress 505