Donkey anti mouse immunoglobulin g igg

We fixed iMNs with 4% paraformaldehyde and blocked them with 5% donkey serum with 0.1% Triton X-100 in 1X PBS. We incubated the cells overnight at 4 °C with the following primary antibodies: anti-SMI32 (mouse monoclonal, 1:1000, BioLegend, cat. no. SMI-32R) and anti-ISL1 (goat polyclonal, 1:250, R&D Systems, cat. no. AF1837). We subsequently rinsed the cells and incubated them with species-specific Alexa Fluor 488-conjugated secondary antibody (donkey anti-mouse immunoglobulin G (IgG), 1:1000, Life Technologies, cat. no. A-21202) and Alexa Fluor 594-conjugated secondary antibody (donkey anti-goat IgG, 1:1000, Life Technologies, cat. no. A-11058). We counterstained nuclei using DAPI (1 μg/mL). We acquired the images using Nikon/Leica microscopes with 10x magnification.

The iPSCs were stained using an Alkaline Phosphatase Staining Kit (StemTAG, cat# CBA-300) and PSC Immunocytochemistry Kit (Invitrogen, cat# A24881) by following the manufacturer’s protocol. NSC cells were stained using the Human Neural Stem Cell Immunocytochemistry Kit (Invitrogen, cat# A24354) following the manufacturer’s protocol. MNP and MN cells were fixed with 4% PFA at room temperature for 15 min or with cold methanol for 5 min, blocked with 10% donkey serum and 0.1% Triton X-100 in 1× DPBS for 1 h. The cells were followed incubated with the primary antibodies (Supplementary Table S3) for 2 h at r.t., washed three times with DPBS containing 0.1% BSA, and incubated with species-specific Alexa Fluor 488-conjugated secondary antibody (donkey anti-mouse immunoglobulin G (IgG), 1:500, Life Technologies) or Alexa Fluor 555-conjugated secondary antibody (Rabbit anti-goat IgG, 1:500, Life Technologies) for 30 min. Cells were then washed three times with DPBS and nuclei stained using Hochest (10 ng/mL) for 10 min. Cell images were acquired using the EVOS FL Auto 2 Imaging System (Invitrogen, cat# AMAFD2000).