Anti pax6

Immunohistochemistry was performed as described previously with slight modifications (Kawasaki et al., 2000 (link); Toda et al., 2013 (link)). Sections were permeabilized with 0.3% Triton X-100 in PBS and blocked with 2% bovine serum albumin (BSA), 0.3% Triton X-100 in PBS. For quadruple-staining and immunostaining with Ki-67, sections were subjected to microwave antigen retrieval in a sodium citrate solution. The sections were then incubated overnight with primary antibodies, which included anti-Hop (Santa Cruz Biotechnology, RRID:AB_2687966), anti-HopX (Atlas Antibodies, RRID:AB_10603770), anti-Tbr2 (R&D Systems, RRID:AB_10569705; Abcam, RRID:AB_778267), anti-Pax6 (Millipore, RRID:AB_1587367), anti-Ki-67 (Thermo Fisher Scientific, RRID:AB_10853185), anti-cleaved caspase 3 (BD Pharmingen, RRID:AB_397274), anti-Brn2 (Santa Cruz Biotechnology, RRID:AB_2167385), anti-Ctip2 (Abcam, RRID:AB_2064130), and anti-GFP antibodies (Nacalai Tesque, RRID:AB_2313652; Medical and Biological Laboratories, RRID:AB_591819). After incubation with secondary antibodies and Hoechst 33342, the sections were washed and mounted.

Aliquots of permeabilised and fixed cells suspended in fluorescence activated cell sorting (FACS) buffer were reacted with Hoechst 33342 (Thermo Fisher Scientific, Perth, United Kingdom; Cat# 1391095). Primary antibodies used were: anti-Pax6 (1:200, rabbit, Millipore, Livingston, United Kingdom; Cat# AB2237); anti-Tuj1 (1:800, mouse, Cambridge Bioscience, Cambridge, United Kingdom; Cat# MMS 435P); anti-PH3 (1:200, mouse, Cell Signaling Technology, Danvers, Massachusetts, United States; Cat# 9706); anti-Ki67 (1:500, rat, eBioscience, San Diego, CA, United States; Cat# 12-5698); anti-Cdk6 (1:500, rabbit, Santa Cruz Biotechnology, Dallas, TX, United States; Cat# SC-177). We used secondary-only and fluorescence-minus-one controls to set thresholds for primary antibody-specific staining. Cells were analyzed using an LSRII flow cytometer and FlowJo V10 software (BD Biosciences, Franklin Lakes, NJ, United States). In all of our experiments, we used the same gate for forward scatter for both wild-type and mutant progenitor cells. Since forward scatter values in flow cytometry are directly linked to cell size, this indicates that the wild-type and mutant cells were in the same size range.

Reverse-transcription PCR (RT-PCR) was performed to detect the expression of endogenous pluripotency genes according to the manufacturer's instructions (TaKaRa, Japan). The primers were as follows: Oct4, 5′-GACAGGGGGAGGGGAGGAGCTAGG-3′ and 5′-CTTCCCTCCAACCAGTTGCCCCAAAC-3′; Nanog, 5′-CAGCCCCGATTCTTCCACCAGTCC-3′ and 5′-CGGAAGATTCCCAGTCGGGTTCACC-3′. AP staining and immunofluorescence analysis were performed as previously described [14 (link)]. The primary antibodies used were as follows: anti-SSEA-3 (1 : 100; Sigma, USA), anti-TRA1-60 (1 : 200; Sigma), anti-TRA1-81 (1 : 500; Sigma), anti-alpha-fetoprotein (AFP; 1 : 500; Sigma), anti-Nestin (1 : 100; Abcam, England), anti-PAX6 (1 : 200; Sigma), anti-NeuN (1 : 100; Abcam), beta-tubulin III (1 : 100; Abcam), and anti-smooth muscle actin (SMA; 1 : 500; Sigma).

NSCs (6.5 × 10⁴ cells/cm²) were grown on poly-ornithine/laminin-coated coverslips and differentiated into dopaminergic progenitors. Cells were fixed in paraformaldehyde (PFA, 4% v/v in PBS) for 20 min and permeabilized with Triton X-100 (0.1% v/v) in PBS for 5 min at room temperature. Cells were then incubated with the following primary antibodies diluted 1:200 in BSA (1% w/v in PBS): anti aS (Abcam), anti-Pax6 (Sigma-Aldrich), anti-Nestin (Abcam), anti-Lmx1A (Abcam), and anti-FOXA2 (Abcam), with a secondary fluorescein isothiocyanate-conjugated goat anti-rabbit immunoglobulin G antibody (DBA; dilution: 1:500) or a secondary Alexa Fluor 568-conjugated goat anti-rabbit (ThermoFisher Scientific; dilution: 1:500). The cells were observed with a confocal microscope (Leica, Buffalo Grove, IL, USA).