*2.6. SDS-PAGE and Immunoblotting*

The bicinchoninic acid assay was performed to determine protein concentration in each tissue homogenate sample. Testis and epididymis tissue samples were mixed in electrophoresis sample bu ffer supplemented with 100 mM DTT, incubated at 95 ◦C for 5 min, and then centrifuged at 21,000× *g* for 5 min. Proteins in the supernatant were electrophoresed on 12% polyacrylamide gels and electrotransfered to polyvinylidene difluoride membranes. Then, the membranes were incubated in a solution of skim milk (5%, *w*/*v*) in Tween-containing Tris-bu ffered saline (TTBS; 20 mM Tris, 0.1% *v*/*v* Tween, pH 7.8) for 30 min followed by the incubation in anti-PRDX-1 (1:10,000), anti-PRDX-6 (1:10,000), anti-catalase (1:1000), anti-4-hydroxynonenal (4HNE) (1:100), anti-Thioredoxin1 (TRX1) (1:500) primary antibodies overnight. To test the specificity, 0.4 μg/mL of anti-PRDX1 was incubated with 2 μg/mL of its antigenic peptide in TBS-T supplemented with 3% BSA for 2 h at room temperature [18]. The absence of non-specific binding was confirmed by the incubation of tissue samples with the secondary antibody (goat anti-mouse or donkey anti-rabbit IgG) only. After being washed with TTBS, the membranes were incubated with goa<sup>t</sup> anti-mouse or donkey anti-rabbit IgG conjugated with horseradish peroxidase (diluted 1:2000 in TTBS) for 45 min at room temperature and washed again with TTBS. The immunoreactive bands were detected using Lumi-Light chemiluminescence kit. Then, the membranes were stripped and re-blotted with an anti-tubulin antibody to determine equal loading. Silver staining was used to determine equal loading in samples under non-reducing conditions. The membrane detection was done by using both Amersham Imager 600 (Thermo Fisher Scientific, Inc., Toronto, ON, Canada) and autoradiography films. The digital images were analyzed using Image J win-64 software (University of Wisconsin-Madison, Madison, WI, USA). The band intensities of the protein were normalized to that of the tubulin to compare the level of expression of the protein of interest.
