Immunocytochemical analysis of Ob and Ob-R proteins expression. Representative confocal images of embryos obtained by ICSI in oocytes matured in vitro, stained with antibodies directed against Ob-R and Ob protein. For each raw, corresponding bright-field (A, B, C, D, E), confocal (A1, B1, C1, D1, E1 for Ob-R; A2, B2, C2, D2, E2 for Ob labeling and A3, B3, C3, D3, E3 for Ob-R and Ob merge) and UV light images of blastomere nuclei (A4, B4, C4, D4, E4) of the same embryo are shown. After Hoechst 33258 staining, to evaluate chromatin configuration, nuclei of regular morphology in each blastomere and residual polar bodies are visible. The 2-cell stage embryo in raw A is representative of embryos issuing from oocytes cultured in presence of 1000 ng/ml leptin; the 4-cell stage embryo in raw B was cultured in presence of 1 ng/ml leptin and the 8-cell stage embryo in raw C was cultured in presence of 10 ng/ml leptin; the 4-cell stage embryo in raw D was cultured in absence of leptin; the 4-cell stage embryo in raw E represents the negative control (no primary antibodies against Ob and Ob-R). Scale bar represent 60 μm.