Secondary PCR was achieved with modified nucleotide digoxigenine-11-dUTP (DIG) (Boehringer Mannheim, Indianapolis, Indiana, USA) as label, with the following pairs of primers: 5′-ccc ttc cga agt ttc tgg cag cag (forward) and 5′-ggg ctc ctc caa ggt ggt gcc c (reverse) for NOS-II or 5′-tcc agt aac aca gac agt gca (forward) and 5′-cag gaa gta agt gag agc (reverse) for NOS-III . In agarose gel electrophoresis stained with ethydium bromide (Sigma), PCR products produced a single band corresponding to the 473 base pair fragment of rat NOS-II or the 693 base pair fragment of rat NOS-III . 18S RNA probes were DIG labelled using a random primed DNA labelling Kit (Boehringer Mannheim). buy ortho tri-cyclen online
NOS-III, NOS-II and beta-actin mRNA expression: Messenger RNA synthesis was studied by means of dot and Northern blot hybridization techniques, and RT-PCR . Total RNA was isolated from freshly excised tissues homogenized in Trizol reagent (Gibco BRL, Life Technologies, Rockville, Maryland, USA) by the method described by Chomczynski , Chomczynski and Mackey , and the manufacturer’s protocol. RNA concentration and purity were estimated from the optical density measured at 260/280 nm and by agarose gel electrophoresis in denaturing conditions followed by digital photography and analysis with the Electrophoresis, Analysis and Documentation System (DC40, Kodak, New Haven, Connecticut, USA). RNA was diluted in RNAse-free water and kept deep frozen (-80°C) until the time of determination of mRNA expression for genes of interest.