*3.3. Lipid Peroxidation*

Lipid peroxidation was evaluated by immunostaining of 4-hydroxynonenal (4-HNE) protein adducts, a by-product of lipid peroxidation. Longitudinal sections of the epididymides of 18-month-old WT and *Sod1*−/− mice revealed major differences in 4-HNE staining intensity and its regional distribution (Figure 3). Staining was absent in the initial segmen<sup>t</sup> and the caput epididymidis and was found almost exclusively in the corpus and the cauda epididymides. Further, in 18-month-old *Sod1*−/− mice, it appeared to be higher in the distal than in the proximal cauda epididymidis. The 4-HNE staining was detected mainly on cytoplasmic membranes, particularly on the apical membranes; in highly stained

cells, it was also visible on intracellular and nuclear membranes. Quantitative analyses were done for staining on all cell surfaces. In the caput epididymidis, staining was indistinguishable to that seen in the control sections without primary antibody. The 3-way ANOVA analysis on quantitative data clearly indicated that there was a significant difference between the WT and *Sod1*−/− genotypes and that this effect was amplified by the age of the mice (Table 2).

**Figure 3.** Lipid peroxidation as assessed by 4-HNE immunostaining is increased in the corpus and cauda epididymides with aging and this phenotype is enhanced in the absence of SOD1 expression. Representative pictures of whole epididymis sections of 18-month-old wild-type and *Sod1*−/−mice after the immunostaining of the 4-HNE (yellow) and the counterstaining of the nucleus (DAPI, blue). Scale bar: 2 mm. Observations have been carried out in epididymis sections of 3-month-old and 18-month-old wildtype and *Sod1*−/−mice. Staining was strongest in the corpus and cauda epididymides on the apical membrane of epithelial cells (arrow) and on intracellular membranes (arrow head). The negative controls (no primary antibody) for immunostaining are displayed for the corpus and cauda epididymides. Scale bar: 40 μm. The 4-HNE staining has been quantified in the epididymal epithelium (clear histograms) and in the interstitial tissue (dashed histograms) separately. \* *p* < 0.05; \*\* *p* < 0.01 (3-way ANOVA, n = 4–5).
