Tcs sp5 sted cw confocal microscope

Coverslips covered by differentiating hiPSC-derived cells were fixed in 2% Paraformaldehyde (PFA) for 20 min at room temperature, followed by several washes with phosphate-buffered saline (PBS). After blocking for 30 min at room temperature with 2% bovine serum albumin (BSA) in PBS, coverslips were incubated in primary antibody overnight at 4 °C. The following primary antibodies were used: guinea pig anti-insulin (1/500, A056401-2, Dako, Glostrup, Denmark) and rabbit anti-NRF2 (1/250, SAB4501984-100UG, Sigma-Aldrich, St. Louis, MO, USA,). After brief washes in PBS, the coverslips were incubated for 3 h at room temperature, in the dark, with the following secondary antibodies: goat anti-guinea pig A488 and donkey anti-rabbit A647 (1/500, Molecular Probes, Eugene, OR, USA). The nuclei were stained with DAPI (D1306, Molecular Probes, Eugene, OR, USA). The coverslips were mounted on glass slides using Prolong Diamond Antifade Mountant Media (P36970, Life technologies, Carlsbad, CA, USA) and images were acquired using a Leica TCS SP5 STED CW confocal microscope (Leica Microsystems, Wetzlar, Germany).

Tissue sections were deparaffinized with xylene and then hydrated through a graded series of ethanol. Antigen retrieval was performed with a heated citric acid-based solution (#H-3300, Vector Laboratories, Burlington, CA, USA). Blocking was performed for 1 h in 10% donkey serum before overnight incubation in primary antibodies (Nephrin and WT1) at 4 °C. The next day, the corresponding fluorescent-labeled secondary antibodies in 10% donkey serum were added for 2 h. Imaging was conducted using a Leica DM 6000B microscope or Leica TCS SP5 STED CW confocal microscope (Leica Microsystems Inc., Buffalo Grove, IL, USA).

Cells were fixed in 2% paraformaldehyde for 30 min before quenching with 0.2 M NH₄Cl for 10 min and permeabilization with 10% BSA in 0.1% Triton X-100 for 10 min. In a humidified chamber, cells were incubated overnight in primary antibody at 4 °C. Cells were incubated in fluorescent-labeled secondary antibody for 1 h at 4 °C. Imaging was performed by using a Leica TCS SP5 STED CW confocal microscope (Leica Microsystems Inc., Buffalo Grove, IL, USA).