Las suite v2

Indirect immunofluorescence against HLA-G in WJ-MSCs obtained from non-vitrified (n = 5) and vitrified (n = 5) WJ tissue samples was performed. An average of 1 × 10⁴ WJ-MSCs were seeded on culture slides (Sigma-Aldrich, Darmstadt, Germany), followed by addition of 1 mL of standard culture medium. After 10 days, the culture slides were microscopically checked and when confluency observed, the indirect immunofluorescence was performed. For this purpose, the WJ-MSCs were fixed for 10 min with 10% v/v neutral formalin buffer (Sigma-Aldrich, Darmstadt, Germany). Antigen retrieval and blocking of cells was applied in all samples, followed by addition of monoclonal antibody against human HLA-G (1:1000, Catalog MA1-10359, ThermoFisher Scientific, Waltham, MA, USA). Secondary FITC-conjugated mouse IgG antibody (1:100, Sigma-Aldrich, Darmstadt, Germany) was added. Cell nuclei became evident with DAPI staining (ThermoFisher Scientific, Waltham, MA, USA). Finally, the slides were glycerol mounted and observed under fluorescent microscope. Images were acquired with LEICA SP5 II microscope equipped with LAS Suite v2 software (Leica, Microsystems, Wetzlar, Germany).