Sperm cells were washed again and observed with confocal microscopy (Nikon AR1 laser confocal scanning microscope coupled to the NIS-Element Software, Florence, Italy)
Sperm cells were washed again and observed with confocal microscopy (Nikon AR1 laser confocal scanning microscope coupled to the NIS-Element Software, Florence, Italy). 3.5. Nr4a3 Philadelphia, PA, USA). Protein content was determined using a MicroBCA? Protein Assay Kit (ThermoFisher) and read at EnSpire Multimode Plate Reader (PerkinElmer, Waltham, MA, USA). Equal amounts of protein per lane (20 g) were loaded on an SDS-PAGE 4C15% gradient gel (Mini-PROTEAN? TGX? Precast Protein Gels, BioRad) and blotted on a nitrocellulose membrane using the Trans-Blot? TurboTM Transfer System (BioRad). Membranes a5IA were blocked in EveryBlot Blocking Buffer (BioRad) for 5 min at room temperature (RT) and then incubated overnight at 4 C with primary antibodies to ACE2 (1 g/mL for ab15348, Abcam and 1:1000 for Novus, NBP2-67692, followed by HRP-conjugated secondary antibodies to rabbit IgG (1:10,000, Santa Cruz Biotechnology, Santa Cruz, CA, USA). Bound antibodies were detected using SuperSignal? West Pico PLUS Chemiluminescent Substrate (ThermoFisher) with the image digitally captured using an Azure C400 (Chemiluminescent Western Blot Imaging System, Azure Biosystems, Dublin, CA, USA). After antibody detection, blots were stripped using a Restore? Western Blot Stripping Buffer (Thermo Fisher) for 30 min at room temperature and re-incubated following the described protocol. Total labelled protein amount detected by Ponceau S was used as loading control as previously a5IA reported (Supplementary Figure S2) [36]. Blots were cut prior to hybridization, never after. Multiple exposure images with full-length membranes and membrane edges were added to the Supplementary Figures S3 and S4. 3.4. Immunocytochemistry Spermatozoa were fixed and permeabilized with a solution containing 4% paraformaldehyde and 0.5% Tryton X-100 for 20 min at RT. Sperm cells were then washed three times with PBS, centrifuging for 5 min at 500 em g /em . a5IA The pellet was resuspended and a volume of 20 L was added to a5IA glass slides, inside a 1cm diameter circles. Once the slides were dry, bovine serum albumin (BSA) 1% was added as a blocking agent for the sperm cells for 30 min at RT within a dry chamber. After washing, spermatozoa were incubated overnight at 4 C with the primary antibody (Abcam ab15348 at 20 g/mL and Novus NBP2-67692 0.5 L in 50 L). After washing three times, a secondary antibody was added as follows: anti-rabbit FITC conjugated for Abcam ab15348 and Novus a5IA SN0754, at a dilution 1:250 incubated for 1 h at RT. To stain the nuclei, 4,6-diamidino-2-phenylindole (DAPI) staining was added during the last 15 min of incubation with the secondary antibodies. Sperm cells were washed again and observed with confocal microscopy (Nikon AR1 laser confocal scanning microscope coupled to the NIS-Element Software, Florence, Italy). 3.5. Flow Cytometry Viable and motile sperm suspensions obtained by swim-up procedure were incubated for 60 min at 37 C with the primary goat anti-ACE2 antibody (R&D Systems, AF933) (0.25 g/106 cells). A non-specific serum from a non-immunized goat, at the same concentration of the primary antibody, was used as a negative control. After washing three times with PBS, spermatozoa were incubated with rabbit anti-goat Cy3 conjugated (1:1000) for 60 min at 37 C. After washing three times, samples had been examined at Becton Dickinson FACSCalibur (Rome, Italy). At least 2 104 occasions had been acquired and examined in each test using Cell Goal software program (San Jose, CA, USA). The percentage of positive spermatozoa as well as the peak route, portrayed as AU, had been examined. 4. Conclusions To conclude, from the tests discussed here, it’s been showed that full-length ACE2 and brief ACE2 proteins are portrayed by mature spermatozoa after ejaculations, highly confirmed using the three-antibody technique in conjunction with cytofluorimetry and immunofluorescence analysis. The demo of receptor proteins expression represents an integral premise for learning the possible influence of SARS-CoV-2 an infection on sperm biology. Acknowledgments Writers recognize all of the volunteers who all participated within this research sincerely. Supplementary Materials The next supporting information could be downloaded at: https://www.mdpi.com/article/10.3390/ijms23073694/s1. Just click here for extra data document.(434K, zip) Writer Contributions All writers contributed extensively to the task presented within this paper. Particularly, N.B. conceived the ongoing work; N.B., M.R.-S. and A.B. designed the tests; B.B. funded and supervised the task; M.R.-S., A.T. and C.C. (Costanza Cimini) performed traditional western blot.