Bcl 2

The muscle biopsies were mechanically pulverized and protein extraction was performed as previously described (Mancini et al., 2017 (link)). Briefly, protein samples (50 μg each) were separated on 4–20% precast gradient polyacrylamide gels (Bio-Rad), transferred to the Hybond ECL nitrocellulose membrane (GE Healthcare) and checked by Ponceau S staining to verify equal loading. The membranes were immunoblotted using rabbit polyclonal antibodies against autophagy related 5 homolog (ATG5), autophagy related 12 homolog (ATG12), mouse monoclonal antibodies against Heat Shock Protein 90 (HSP90), Heat Shock Protein 70 (HSP70), B-cell lymphoma 2 (Bcl-2) (Elabscience, 1:500), monoclonal rabbit antibody proteasome 26S subunit non-ATPase 13 (PSMD13) (Abcam, 1:1000), monoclonal mouse antibody against glyceraldehyde- 3-phosphate dehydrogenase (GAPDH) (1:1000; Santa-Cruz Biotechnology Inc.). Blots were incubated with appropriate horseradish peroxidase-conjugated secondary antibody and target proteins were visualized by ECL detection (GE Healthcare). Densitometric measurements were carried out using Quantity One software (Bio-Rad) as reported elsewhere (Imperlini et al., 2015 (link)). GAPDH protein was used to estimate the total amount of loaded proteins. Results were normalized as a percentage of the mean of controls in each membrane.

The liver paraffin sections were dewaxed and processed as previously described [34 (link),36 (link)]. Sections were IHC stained using the primary antibodies: rabbit-polyclonal-antibody against NF-kB p65 (1:200), Nrf2 (1:200) (Fisher-Scientific Inc., Waltham, MA, USA), Bcl2 (1:200), and caspase-3 (1:200) (Elabscience Biotechnology Inc., Houston, TX, USA). Diaminobenzidine (DAB) was used for visualization.

The efficacy of OME on the expression of the Bax and Bcl-2 protein was evaluated by immunohistochemical technique [22 (link)]. Briefly, the tissue sections were deparaffinized and rehydrated and then immersed in sodium citrate buffer (10 mM) for 10 min, restoring their antigenicity. Tissue specimens were put in Tris-buffered saline for cooling before applying the ARK peroxidase kit (DAKO Denmark A/S, Glostrup, Denmark). The blocking of endogenous peroxidase was carried out by exposing them to the peroxidase solution for 5 min and then the samples were rinsed. The slides were incubated with biotinylated primary antibodies against Bax (1:100) and Bcl-2 (1:100) (Elabscience, Huston, TX, USA) for fifteen minutes and then treated with (exposed to) streptavidin-HRP for 30 min. The slides were stained with diaminobenzidine substrate chromogen and hematoxylin.

For hematoxylin and eosin (H&E) stain, liver and kidney tissues were fixed in neutral buffered formalin, ethanol dehydrated, and paraffin embedded. Paraffin blocks were cut and stained with H&E. Sections were randomly examined in a blind manner and the pathological injuries were graded as previously described [30 (link),31 ,32 (link)].
For IHC, paraffin sections of the liver and kidney were dewaxed and handled as previously demonstrated [30 (link),33 (link)]. IHC was carried out using the primary antibodies: Nrf2 (1:200) (Fisher Scientific Inc., Waltham, MA, USA), rabbit-polyclonal-antibody against NF-κB p65 (1:200), caspase-3 (1:200), and Bcl2 (1:200) (Elabscience Biotechnology Inc., Houston, TX, USA). Diaminobenzidine (DAB) was utilized for visualization.