Dab tablet

Immunohistochemistry experiments were carried out as previously described (Gao et al., 2018 (link)). Briefly, after the Thy1-YFPH mice were anesthetized, the eyes were enucleated on P21 and fixed in 4% paraformaldehyde (PFA) for 30 min. The retinas were isolated from eyeballs, fixed in 4% PFA for additional 10 min, and incubated with 3% H₂O₂ for 20 min. After being blocked in a blocking solution (5% normal donkey serum plus 1% BSA, 0.2% glycine, 0.2% lysine, and 0.3% Triton X-100) for 2 h at room temperature, retinas were incubated with goat polyclonal antibodies against GFP (1:500, NB100-1770, Novus Biologicals) for 2 days at 4°C. Then the retinas were sequentially incubated with the biotinylated donkey anti-goat antibodies, the avidin-biotin complex (Vectastain ABC Elite Kit; Vector Laboratories, USA), the 3,3′-diaminobenzidine (DAB tablets, Sigma), and finally flat-mounted on glass slides with aqueous mounting medium (IMMCO Diagnostics, Inc).

Rats were deeply anesthetized with isoflurane and transcardially perfused. Immunohistochemistry in brain slices was also performed as described in Salti et al. (2015 (link)). Briefly, the sections were permeabilized, blocked and then incubated with a rabbit monoclonal SATB2 antibody (1:200, abcam #ab 29446). The sections were subsequently incubated with the secondary biotinylated anti-rabbit antibody (1:200, Vector Laboratories #BA-1000), then with avidin-biotinylated horseradish peroxidase complex (ABC Elite kit) and finally in in 3,3-diaminobenzidine tetrahydrochloride (DAB tablets, Sigma). Afterward the sections were mounted onto Leica Extra adhesive micro slides and allowed to dry before coverslipping. For each brain region, three sections per animal were scanned, using a Zeiss optical microscope set at ×20 magnification equipped with a camera (Axioplan 2 Imaging) interfaced to a PC. Intensity of DAB staining as described by Nguyen et al. (2013 (link)) was evaluated by an experimenter blind to treatment conditions using Fiji imaging software. The mean of the intensity value of the three sections/region/animal were included in the statistical analysis.

iHP cells or single colonies (5 × 10⁴) from in CFC assay were suspended in 100 μl of 10% FBS in PBS and cytospined to the slides. The slides were fixed with cold methanol for 2 min and allowed to dry for 1 h. Then slides were stained using DAB tablets (3, 3′-Diaminobenzidine tetrahydrochloride, Sigma), followed by stained with Wright–Giemsa solution (Sigma) according to the instruction of products. The slides were allowed to dry overnight, mounted with mounting medium and photographed under microscope (Leica DM LB2).