Anti fus antibody pa5 52610

Sperm cells collected from caput/cauda epididymis of CTRL (n = 4) and HFD (n = 4) mice, dried on slides as reported above, were used for immunofluorescence analysis. In detail, sperm cells were fixed with a fix solution consisting of 4% paraformaldehyde (sc-281692; Santa Cruz Biotechnology, Heidelberg, Germany) for 20 min at RT. Then, a permeabilization step was carried out by using 0.1% Triton X-100 (X100; Sigma-Aldrich, Milano, Italy). Blocking was conducted with 10% of donkey serum (ab7475; Abcam, Cambridge, UK) for 30 min at RT, and then sperm cells were incubated with anti-FUS antibody (PA5-52610; Invitrogen, Milano, Italy) overnight at 4 °C. A negative control consisting of primary antibody omission and an isotype control by using the same isotype (IgG) at the same concentration of FUS primary antibody (rabbit IgG, polyclonal isotype control (ab37415) from Abcam, Cambridge, UK) were carried out (Figure S6). Slides were washed 3 times in Dulbecco’s PBS (DPBS, 1X) until the addition of Texas Red conjugated antibody (Jackson ImmunoResearch, Cambridge, UK) for 1 h at 37 °C. Nuclei visualization was performed, incubating the slides with DAPI solution (D9542; Sigma-Aldrich, Milano, Italy). Immunofluorescence analysis was conducted under an optical microscope (Leica DM 5000 B + CTR 5000) with a UV lamp.

SPZ collected from caput/cauda epididymis of CTRL, HFD and FS mice (n=4 for each experimental group) and cauda SPZ of CTRL mice in vitro treated with vehicle or H₂O₂ were used for immunofluorescence analysis. Samples were fixed in 4% paraformaldehyde (sc-281692; Santa Cruz Biotechnology, Heidelberg, Germany) for 20 min at RT. Permeabilization step was conducted with 0.1% Triton X-100 (X100; Sigma-Aldrich, Milano, Italy), while blocking was conducted with 10% of donkey serum (ab7475; Abcam, Cambridge, UK) for 30 min at RT. Slides were then incubated with different primary antibodies [anti-FUS antibody (PA5-52610; Invitrogen, Milano, Italy); anti-4HNE antibody (ab46545; Abcam, USA)] overnight at 4°C. Following three washes in Dulbecco’s PBS (DPBS, 1X), a conjugated secondary antibody was used (Jackson ImmunoResearch, Cambridge, UK) for 1 h at 37°C. Nuclei were labeled with DAPI (D9542; Sigma-Aldrich, Milano, Italy) and slides were observed under an optical microscope (Leica DM 5000 B + CTR 5000) with a UV lamp.