Ab75639

Immunohistochemistry was performed as previously described (9 (link)). Briefly, sections of all groups were incubated overnight at 4°C with the following primary antibodies: p-eNOS (Abcam, ab75639, 1:50), mTOR (Abcam, ab2732, 1:1000), Beclin-1 (Abcam, ab62557, 1:100), ATG5 (Abcam, ab228668, 1:100) and iNOS (Abcam, ab3523, 1:100. The labeling reaction was performed in six sections per group and the analysis of pixels quantification was performed in 6-8 images per group (mean value). Images of the same magnification were quantitatively analyzed using Gimp 2.6 software (GNU Image Manipulation Program, UNIX platforms). Unspecific labeling/background was removed from the quantification by using the selection and exclusion tool of the aforementioned software.

The tissue expression of iNOS, eNOS, and phosphorylated eNOS (phospho S1177) was determined using western blot. The aliquots of 60 µg of total protein from heart homogenates were separated on 12% SDS-PAGE. Then, iNOS, eNOS, and phospho-eNOS proteins were transferred on PVDF membranes (Bio-Rad) and were detected with primary antibody (mouse anti-iNOS polyclonal antibody 1 : 5000 (Abcam, ab21775); anti-eNOS (Abcam, ab50010); anti-phospho (S1177)-eNOS (Abcam, ab75639), respectively) and secondary goat anti-mouse IgG horseradish peroxidase conjugate 1 : 1000 (Bio-Rad, STAR207P). Clarity^TM Western ECL (Bio-Rad) substrate was used for proteins detection. ChemiDoc^TM MP System and Quantity One Software (Bio-Rad) were used for detection of bands and measurement of their density. iNOS, eNOS, and phospho-eNOS quantities were expressed as AU normalized to total protein amount.