Horseradish peroxidase conjugated goat anti rabbit and rabbit anti goat secondary antibodies

We followed the methods of Manchini et al. [32 (link)] and Melo et al. [33 (link)] for the muscle tissue processing and for protein extraction. Protein samples (20 μg) were subjected to SDS-PAGE in 10–12% polyacrylamide gel. Separated proteins were transferred onto hydrophobic membranes (Hybond-P, Amersham Biosciences; Piscataway, NJ, USA), in which they were soaked in a blocking buffer (5% nonfat dry milk and 0.1% Tween 20 in PBS, pH 7.5) for 60 min at room temperature and then incubated overnight at 4°C with primary antibodies: rabbit anti-4-HNE (1 : 2000 dilution; Abcam, Cambridge, MA, USA); rabbit anti-SOD1 (1 : 5000 dilution; Abcam, Cambridge, MA, USA); rabbit anti-CAT (1 : 5000 dilution; Abcam, Cambridge, USA); goat anti-HSP70 (1 : 1000; Abcam, Cambridge, MA, USA); and anti-GAPDH (1 : 500; Santa Cruz Biotechnology, Santa Cruz, CA, USA). Then, membranes were washed five times and incubated for 60 min with horseradish peroxidase-conjugated goat anti-rabbit and rabbit anti-goat secondary antibodies (1 : 2000; Invitrogen, San Diego, CA, USA). Membranes were washed five times again with blocking buffer and then rinsed twice in PBS buffer. Bound antibody was detected by using chemiluminescence reagent for 60 s. The bands were imaged by using Amersham Imager 600 system (GE Health Care, Little Chalfont, UK, USA).

Proteins were extracted from the LV remote area as previously described by us (Silva et al., 2014 (link)). Homogenate protein samples of 30 μg were subjected to SDS-PAGE in 10% polyacrylamide gel. Separated proteins were transferred onto hydrophobic polyvinylidene difluoride membranes (Hybond-P, Amersham Biosciences; Piscataway, J, USA), and the transfer efficiency was examined with 0.5% Ponceau S. The membranes were soaked in a blocking buffer (5% nonfat dry milk and 0.1% Tween 20 in PBS, pH 7.5) for 1 h at room temperature and then incubated overnight at 4°C with primary antibodies: rabbit anti-Akt1 (1:5000 dilution; Abcam, Cambridge, MA, USA); rabbit anti-phosphoSer473Akt1 (1:5000 dilution; Abcam, Cambridge, USA); goat anti-VEGF (1:1000; Abcam, Cambridge, MA, USA); anti-GAPDH (1:500; Santa Cruz Biotechnology, Santa Cruz, CA, USA). After overnight incubation, membranes were washed five times and then incubated for 1 h with horseradish peroxidase-conjugated goat anti-rabbit and rabbit anti-goat secondary antibodies (1:2000; Invitrogen, San Diego, CA, USA). Membranes were finally washed five times with blocking buffer and then rinsed twice in PBS. Bound antibody was detected by using chemiluminescence reagent for 1 min. The bands were imaged by using Amersham Imager 600 system (GE Health Care, Little Chalfont, UK). UK).