Rabbit anti islet1

On the 5th DIV, cells were incubated for 1 h with the MitoTracker stain (MitoTracker™ Red CMXRos) mixed with neurobasal medium 1:100 nM und washed once with 1× PBS. Cells were fixed in 4% paraformaldehyde for 20 min at RT. Afterward, wells were washed thrice with 1× PBS. Cells were blocked in blocking buffer consisting of 0.2% Triton-X-100 (Sigma), 10% goat serum (Invitrogen), and 2.5% BSA at RT for 30 min. After blocking, primary antibodies were added (rabbit anti-islet1 [1:500; Abcam]) in blocking solution and incubated overnight at 4 °C. The next day, wells were washed thrice with 1× PBS. Secondary antibodies (AlexaFluor 555 goat anti-rabbit [1:1000; Invitrogen]) were diluted in blocking buffer and incubated for 2 h at RT. Cells were washed thrice with 1× PBS and once with distilled water. After washing was completed, cells were mounted in mounting medium (Mowiol; Roth, Karlsruhe, Germany) containing 0.1% DAPI (Sigma), and visualization took place through fluorescence microscopy (BX61; Olympus Deutschland GmbH, Hamburg, Germany). Image acquisition was performed with Cell F and CellSens Dimensions Ink. Software 1.18 (Build 16686, Olympus Deutschland GmbH, Hamburg, Germany), using a 20× objective and a numerical aperture of 0.17.

To assess the purity of the cells, we performed immunofluorescence studies. All the steps, unless otherwise indicated, were performed at room temperature. Cultured cells were fixed in 4% buffered paraformaldehyde (PFA) for 10 minutes and then washed in PBS three times. After a common permeabilization step in 0.2% Triton X-100 in PBS for 10 minutes and an additional three washes in PBS, one set of samples was treated as follows: nonspecific binding was blocked by incubation in 10% normal goat serum for 1 hour and then incubated overnight at 4°C in rabbit anti-Islet-1 (Abcam, Cat. # ab20670, 2 μg/ml) or rabbit anti-GATA-4 (Abcam, Cat. # ab61170, dil. 1 : 500). After three additional washes in PBS, samples were incubated for 1 hour in FITC-conjugated goat anti-rabbit IgG antibody (Vector Laboratories, Burlingame, CA, Cat. # FI-1000, 10 μg/ml). Another set of samples was blocked in 10% normal rabbit serum for 1 hour and then incubated in goat anti-Nkx2.5 (Santa Cruz, Cat. # sc-8697, dil. 1 : 50) overnight at 4°C. Then, samples were incubated in AlexaFluor® 488-conjugated rabbit anti-goat antibody (Invitrogen, Cat # A-11078, dil. 1 : 200) for 1 hour. All the samples were then washed three times in PBS and mounted with VECTASHIELD® antifade mounting medium with DAPI for nuclear staining (Vector Laboratories, Cat. # H-1200).

Control and mutant iPSC clones were differentiated into motor neurons as described (31 (link)). In summary, after dissociation of embryoid bodies containing motor neuron progenitors at D10, single cells were plated on poly ornithine-laminin–coated coverslips at 2 × 10⁵ cells per coverslip for 1, 4, and 7 more days. Cells were fixed with 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS) for 10 min at room temperature (RT). For staining, cells were treated with 5% goat serum with 0.1% Triton X-100 (Thermo Fisher Scientific) for 1 hour at RT and then incubated overnight at 4°C with a mouse anti–βIII-Tubulin (TUJ1, Sigma-Aldrich, 1/500) and a rabbit anti-ISLET1 (Abcam, 1/100) antibodies in 5% goat serum. Secondary antibodies [goat anti-mouse IgG2a-Alexa 555 (1/2000) and goat anti-rabbit IgG Alexa 488 (1/2000) from Thermo Fisher Scientific] were incubated for 1 hour at RT with Hoechst33342 to stain nuclei.