Dmi6000 fluorescence microscope

The immunolocalization of the phosphorylated form of AMPK was performed in cryopreserved spermatozoa treated with 100 µM CGA and untreated. Spermatozoa were washed in phosphate buffer saline (PBS), smeared on glass slides, air dried and fixed in 4% paraformaldehyde (PFA) for 15 min. After a treatment with blocking solution (PBS-BSA 1% NGS 5%) for 20 min, slides were incubated overnight at 4 °C with a primary antibody anti-Phospho-AMPKα (Thr172) (Cell Signaling Technology, Danvers, MA, USA) diluted 1:100. The reaction was revealed by an anti-rabbit antibody raised in a goat Alexa Fluor^® 488 conjugate (Invitrogen, Thermo Fisher Scientific, Carlsbad, CA, USA), diluted at 1:100. Incubation without the primary antibodies was used as control. Nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI) solution (Vysis, Downers Grove, IL, USA). Observations were made with a Leica DMI 6000 Fluorescence Microscope (Leica Microsystems, Germany), and the images were acquired by the Leica AF6500 Integrated System for Imaging and Analysis (Leica Microsystems, Germany). The experiments were performed on 8 samples and assessed twice by two different examiners.

p-hIFs were seeded in 12-well plates (4 × 10⁵ cells/well) containing coverslips. After 72 h of different treatments, coverslips were rinsed with PBS, fixed with 4% paraformaldehyde for 10 min, and permeabilized with 0.1% Triton X-100 for 10 min at room temperature. Non-specific antibody binding was blocked with 3% bovine serum albumin/PBS solution for 30 min. Then, coverslips were incubated with primary collagen I antibody (1:1000, 1310-01, Southern Biotech, Birminghan, UK) for 1 h at 37 °C. Afterward, coverslips were rinsed with PBS three times and incubated with Alexa-Fluor488-conjugated rabbit anti-goat secondary antibodies (1:400 A11008; Molecular Probes, Leiden, The Netherlands) for 30 min. Nuclei were stained with Mounting Medium with 4’,6-diamidino-2-phenylindole (DAPI H-1200 Vector Laboratories, Peterborough, UK). Images were taken using a Leica DMI6000 fluorescence microscope (Leica Microsystems GmbH).

Sperm samples were washed in phosphate buffer saline, smeared on glass slides, air dried, and processed as previously reported [31 (link)]. Briefly, the slides were incubated overnight at 4°C with rabbit polyclonal anti-8-iso-PGF_2α antibody (Abcam, Cambridge, UK), diluted at 1 : 100; reaction was revealed by an anti-rabbit FITC conjugate antibody raised in a goat (Sigma-Aldrich, Italy), diluted at 1 : 300. Incubation in the primary antibodies was omitted in the control samples. Slides were mounted with 4,6-diamidino-2-phenylindole (DAPI) solution (Vysis, Downers Grove, IL). Observations were made with a Leica DMI 6000 Fluorescence Microscope (Leica Microsystems, Germany), and the images were acquired by the Leica AF6500 Integrated System for Imaging and Analysis (Leica Microsystems, Germany). At least five hundred sperm from each sample was examined.

Swim-up-selected human sperms, incubated or not with F₄-NeuroPs, were washed in PBS, smeared on glass slides, air-dried, and fixed in 4% paraformaldehyde for 15 min. After treatment with a blocking solution (PBS–bovine serum albumin (BSA) 1% Normal Goat Serum (NGS) 5%) for 20 min, the slides were incubated overnight at 4 °C with a primary antibody anti-Phospho-AMPKα (Thr172) (Cell Signaling Technology, Danvers, MA, USA) diluted at 1:100; reaction was revealed by an anti-rabbit antibody raised in a goat Alexa Fluor^® 488 conjugate (Invitrogen, Thermo Fisher Scientific, Carlsbad, CA, USA), diluted at 1:100. Incubation without the primary antibodies was used as the control. Nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI) solution (Vysis, Downers Grove, IL, USA). Observations were made with a Leica DMI 6000 Fluorescence Microscope (Leica Microsystems, Germany), and images were acquired using the Leica AF6500 Integrated System for Imaging and Analysis (Leica Microsystems, Germany). Two hundred sperms per sample were evaluated. Five samples were analyzed.

Metaphase spreads were prepared as described previously [18 (link)]. In brief, HEK293 cells were incubated with 0.1 µg/mL colcemid for 3 h and harvested by trypsinization. Cell pellets were resuspended in 5 mL of hypotonic potassium chloride (75 mM) solution and incubated for 10 min at 37 °C for swelling. Cells were fixed once with 5% acetic acid for 3 min and then twice with ethanol-acetic acid (3:1) for 10 min. Fixed cells were gently resuspended in fixative solution to yield an optimal cell density and dropped onto glass slides before staining with Giemsa or DAPI. Phase contrast or fluorescent images were acquired using an Olympus IX83 inverted or a Leica DMI 6000 fluorescence microscope, respectively, at 63× magnification.

GFP‐LC3 HEK 293 cells were plated on uncoated 16‐mm glass coverslips at 40 000 cells per well and treated with Z‐VAD‐fmk, Q‐VD‐OPh (50 μm, 24–72 h) or a vehicle control. For genetic ablation of N‐glycanase, HEK 293 cells were transfected with SMARTpool: ON‐TARGETplus NGLY1 siRNA (25 nm) or an ON‐TARGETplus nontargeting control siRNA (25 nm) and analysed 3–5 days post‐transfection. Cells were incubated with FURA‐2 AM (1 μm) in imaging buffer (121 mm NaCl, 5.4 mm KCl, 0.8 mm MgCl₂, 1.8 mm CaCl₂, 6 mm NaHCO₃, 5.5 mm D‐glucose, 25 mm HEPES, pH 7.4 supplemented with Gibco MEM amino acid and MEM nonessential amino acid solution) for 20 min at 37 °C. Cells were washed three times with imaging buffer and incubated for a further 20 min. Coverslips were imaged using a Leica DMI6000 fluorescence microscope on the 20× objective. Ca²⁺ was mobilized from intracellular stores using thapsigargin (1 μm) after 180 s. Images were taken every 20 s for 12 min. Time lapse videos were analysed using imagej [50 (link)]. Cytosolic areas of cells were selected plus a background region for 340 and 380 nm. A minimum of 20 cellular regions were analysed per coverslip. The 340/380 ratio was calculated. The area under the curve, baseline average of the first eight images and peak height were calculated using GraphPad Prism 7 (GraphPad Software, La Jolla, CA, USA, www.graphpad.com).