PMC:4536246 / 6795-8550 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/4536246","sourcedb":"PMC","sourceid":"4536246","source_url":"https://www.ncbi.nlm.nih.gov/pmc/4536246","text":"Western blotting\nRats (N = 6 per experimental group) were perfused transcardially with 250 ml cold saline, 4 h after GTN or vehicle administration. Brains were immediately removed and chopped into parts; brain areas of interest were dissected out and used for the preparation of total extracts. The samples were homogenized on ice with a homogenizer in at least 5 volumes of modified RIPA buffer (Tris 50 mM, pH 7.4, NaCl 150 mM, EDTA 1 mM, SDS 0,2 %) supplemented with cocktail inhibitors protease. Then, they were incubated on ice for 20 min. The tissue lysate was centrifuged at 10,000 × g for 45 min at 4 °C and supernatants stored at −80 °C. Protein assay was performed by bicinchoninic acid (BCA) method. A 20 μg of protein were submitted to SDS-poliacrylamide gels 10 % and transferred onto a PVDF membrane (Amersham Biosciences). After blocking with 5 % dry milk, the blots were probed overnight at 4 C° with rabbit polyclonal anti-nNOS serum (1:1000; Cayman Chemical) or anti-eNOS serum (1:1000; Santa Cruz Bioctenology) and then probed for 1 h with an anti-rabbit horseradish peroxidase coupled secondary antibody (1:10000; Amersham Biosciences). An enhanced chemiluminescence system (ECL Advance; Amersham Biosciences) was used for visualization. Membranes were also probed with a rabbit polyclonal anti-β actin antibody (1:1000; Santa Cruz Biotechnology) as a housekeeping protein.\nFor semiquantitative analysis, a Bio-Rad GS800 densitometer was used. NOS expression was evaluated in each sample by dividing the optical density of the NOS band by the intensity of the optical density of the band corresponding to the housekeeping protein. The specificity of the antibodies was confirmed by immunoprecipitation with a specific blocking peptide.","divisions":[{"label":"title","span":{"begin":0,"end":16}},{"label":"p","span":{"begin":17,"end":1393}}],"tracks":[]}