Journal: Biological Research

Article Title: Inhibition of forward and reverse transport of Ca 2+ via Na + /Ca 2+ exchangers (NCX) prevents sperm capacitation

doi: 10.1186/s40659-024-00535-9

Figure Lengend Snippet: Identification of NCX isoforms. Identification of NCX1, NCX2 and NCX3 in porcine sperm. Representative immunoblots using sperm samples from different boars with A ) anti-NCX1, ( B ) anti-NCX2 and ( C ) anti-NCX3 antibodies, and their respective blocking peptides

Article Snippet: To block nonspecific binding sites, samples were incubated with a blocking solution consisting of 5% BSA in PBS at room temperature for 1 h. Subsequently, sperm were incubated with primary NCX antibodies, either NCX1, NCX2 or NCX3 (Alomone Labs), diluted 1:100 (v: v) at room temperature for 1 h. After washing five times with PBS (5 min per wash), samples were incubated with a secondary anti-rabbit antibody Alexa Fluor Plus 488 (ref. A32731, Invitrogen, Waltham, MA, USA) diluted 1:200 (v: v) in blocking solution at room temperature for 1 h. Samples were again washed five times with PBS (5 min per wash), air dried and mounted with 10 μL of ProLongTM Glass Antifade Mountant with NucBlue (Hoechst 33342; ref. P36985, Invitrogen) in the dark.

Techniques: Western Blot, Blocking Assay

Journal: Biological Research

Article Title: Inhibition of forward and reverse transport of Ca 2+ via Na + /Ca 2+ exchangers (NCX) prevents sperm capacitation

doi: 10.1186/s40659-024-00535-9

Figure Lengend Snippet: Immunolocalization of NCX1 isoform. Localization of NCX1 in the plasma membrane of porcine sperm ( A – C ), and after the peptide competition assay ( D – F ). NCX1 appears stained in green (Alexa Fluor 488) and nuclei in blue (DAPI; 4′6′-diamidion-2-phenylindole). Scale bar: 15 μm

Article Snippet: To block nonspecific binding sites, samples were incubated with a blocking solution consisting of 5% BSA in PBS at room temperature for 1 h. Subsequently, sperm were incubated with primary NCX antibodies, either NCX1, NCX2 or NCX3 (Alomone Labs), diluted 1:100 (v: v) at room temperature for 1 h. After washing five times with PBS (5 min per wash), samples were incubated with a secondary anti-rabbit antibody Alexa Fluor Plus 488 (ref. A32731, Invitrogen, Waltham, MA, USA) diluted 1:200 (v: v) in blocking solution at room temperature for 1 h. Samples were again washed five times with PBS (5 min per wash), air dried and mounted with 10 μL of ProLongTM Glass Antifade Mountant with NucBlue (Hoechst 33342; ref. P36985, Invitrogen) in the dark.

Techniques: Clinical Proteomics, Membrane, Competitive Binding Assay, Staining, Immunofluorescence