Dm 5000 b ctr 5000

For immunofluorescence staining, testis sections were permeabilized with PBS pH 7.4 containing 0.1% Triton-X-100 for 30 min after deparaffinization and rehydration. Antigen retrieval was performed by pressure cooking slides for 3 min in 0.01 M citrate buffer (pH 6.0). Then, non-specific binding sites were blocked with PBS containing 5% BSA and normal goat serum diluted 1:5. Later, sections were incubated with anti-StAR, anti-PCNA, anti-PREP, and anti-α-tubulin antibodies (all diluted 1:100 in the blocking solution) overnight at 4 °C. After three washes in PSB, the appropriate secondary antibody diluted 1:500 in the blocking mixture, was added for 1 hr at RT. Finally, the cells nuclei were marked with Vectashield + DAPI. The sections were observed and captured with the optical microscope (Leica DM 5000 B + CTR 5000) with UV lamp and saved with IM 1000 software. Densitometric analysis of PCNA immunofluorescence was performed with ImageJ Software counting 20 seminiferous tubules/animal for a total of 100 tubules per group.

For DAAM1 co-localization with actin, 5 µm-testis sections were dewaxed, rehydrated and processed as described by Chemek et al. [47 (link)]. Antigen retrieval was performed by pressure cooking slides for 3 min in 0.01 M citrate buffer (pH 6.0). Then, the slides were incubated with 0.1% (v/v) Triton X-100 in PBS for 30 min. Later, nonspecific binding sites were blocked with normal goat serum diluted 1:5 in PBS containing 5% (w/v) bovine serum albumin (BSA; A2153; Sigma–Aldrich, Milan, Italy) before the addition of anti-DAAM1 and anti- β-actin antibody diluted 1:100, for overnight incubation at 4 °C. After washing in PBS, slides were incubated for 1 h with the appropriate secondary antibody (#A-11008; Alexa Fluor 488, Invitrogen; FITC Jackson, ImmunoResearch, Pero MI, Italy; SAB4600082; Anti-Mouse IgG 568, Sigma–Aldrich, Milan, Italy) diluted 1:500 in the blocking mixture. The slides were mounted with Vectashield + DAPI (4′,6-diamidino-2-phenylindole; H-1200-10; Vector Laboratories, Peterborought, UK) for nuclear staining and then observed under an optical microscope (Leica DM 5000 B + CTR 5000). Images where viewed and saved with IM 1000 software. Densitometric analysis of DAAM1 immunofluorescence was performed with ImageJ Software counting 30 seminiferous tubules/animal (five controls and five D-Asp treated rats) for a total of 150 tubules per group.

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.

Cells were permeabilized for 10 min with 0.1% (w/v) Triton X-100 in PBS at room temperature. Later, nonspecific binding sites were blocked with normal goat serum diluted 1:5 in PBS containing 5% (w/v) BSA before the addition of anti-DAAM1 and anti- β-actin antibody diluted 1:100, for overnight incubation at 4 °C. After washing in PBS, slides were incubated for 1 h with the appropriate secondary antibody (#A-11008; Alexa Fluor 488, Invitrogen; FITC Jackson, ImmunoResearch, Pero MI, Italy; SAB4600082; Anti-Mouse IgG 568, Sigma–Aldrich, Milan, Italy) diluted 1:500 in the blocking mixture. The slides were mounted with Vectashield + DAPI (H-1200-10; Vector Laboratories, Peterborought, UK) for nuclear staining and then observed under the optical microscope (Leica DM 5000 B + CTR 5000) and images where viewed and saved with IM 1000 software. We performed two different negative controls: (1) by omitting the primary antibody and (2) by using rat isotype IgG (#I5006, Sigma-Aldrich, Milan, Italy) Densitometric analysis of DAAM1 immunofluorescence was performed with ImageJ Software. Fifty cells, either control and D-Asp treated GC-1, were counted in three different immunofluorescent experiments, for a total of 150 each. Moreover, DAAM1 colocalization analysis with β-actin or nucleus was performed using the Intensity correlation analysis plug-in incorporated into ImageJ.

The fixed slides of colon tissue, were dewaxed, rehydrated and processed. Briefly, antigen retrieval was performed by pressure-cooking slides for 3 min in 0.01 M citrate buffer (pH 6.0). To prevent nonspecific interactions of antibodies, the slides were treated for 2 h in 5% BSA in PBS. Immunostaining was carried out by incubation, overnight at 4 °C, with anti-E-cadherin and anti-β-Catenin antibodies (1:100, Alexa Fluor^®, BD Pharmingen^TM). The slides were mounted on microscope slides by Mowiol + DAPI for nuclear staining, and then observed under the optical microscope (Leica DM 5000 B + CTR 5000).

Apoptosis was examined in paraffin sections by the TUNEL-assay using DeadEnd™ Fluorometric TUNEL System following manufacture’s protocol. Briefly, sections were dewaxed, rehydrated, and washed in 0.85% NaCl and then PBS (13.6 mM NaCl; 2.68 mM KCl; 8.08 mM Na₂HPO₄; 18.4 mM KH₂PO₄; 0.9 mM CaCl₂; 0.5 mM MgCl₂; pH 7.4). To permeabilize the tissues, slides were treated with proteinase K for 10 min at RT. Then, slides were equilibrated in a specific buffer for 10 min at RT, followed by incubation with TdT enzyme and nucleotide mix for 1 h at 37 °C. Reaction was stopped with incubation for 10 min in SSC2X. Finally, the cells nuclei were marked with Vectashield + DAPI. The sections were observed and captured with the optical microscope (Leica DM 5000 B + CTR 5000) with UV lamp and saved with IM 1000 software.

Cauda epididymal SPZ (n = 6 animals for each genotype) samples were dried on slides (n = 3 for each animal). After glutaraldehyde fixation, slides were incubated for 5 min in water, 10 min in 5% aniline blue solution diluted in 4% acetic acid solution, twice for 2 min in water, 2 min in 70, 90, and 100% ethanol solutions and, finally, 2 min in toluene. Slides were then mounted with Eukitt^® and analyzed using a microscope with a transmitted light microscope 100× objective with oil. A total of 100 SPZ for each slide were evaluated, and microscopic analysis was performed under an optical microscope (Leica DM 5000 B + CTR 5000). A minimum of 100 sperm cells were evaluated and counted for each assay.