FIGURE 2.

Co-immunolocalization of 4HNE and proAKAP4 in mouse round spermatids. (A) Following exposure to 4HNE (50 or 100 μM for 1 h), round spermatids were fixed in paraformaldehyde and sequentially labeled with anti-proAKAP4 (red), anti-4HNE (green) and corresponding fluorescently labeled secondary antibodies. Cells were counterstained with the nuclear stain DAPI (blue). (B) Co-localization of target proAKAP4 and 4HNE antigens was confirmed through the application of a proximity ligation assay, whereby fixed round spermatids were incubated with target primary antibodies (anti-proAKAP4 and anti-4HNE) or (C) negative controls (anti-proAKAP4 and anti-androgen receptor; anti-proAKAP4 alone) and oligonucleotide-conjugated secondary antibodies (PLA probes). PLA probes were then ligated and the signal was amplified according to the manufacturer’s instructions (Sigma-Aldrich). The red fluorescent signals generated when target antigens reside within <40 nm were visualized using fluorescence microscopy. Scale bars = 10 μm.