Mouse anti hna

After the prescribed period of graft survival (1 or 6 months), a subset of animals (n = 5/group) were killed by an overdose of sodium pentobarbitone (100 mg/kg), and transcardially perfused with 4% paraformaldehyde and cryosectioned. Immunohistochemistry was performed on fixed cell cultures or brain sections as previously described (Somaa et al., 2017 (link)). Primary antibodies and dilutions were as follows: 4′,6-diamidino-2-phenylindole (DAPI, 1 μg/mL; Sigma Aldrich), goat anti-FOXA2 (1:200; Santa Cruz Biotechnology), chicken anti-GFP (1:1,000; Abcam), rabbit anti-GFAP (1:800; DAKO), mouse anti-HNA (1:300; Millipore), rabbit anti-Ki67 (1:1,000; Thermo Fisher), mouse anti-NESTIN (1:200; Millipore), mouse anti-Neuroligin 3 (Nlgn3, 1:100; Synaptic Systems), mouse anti-OCT4 (1:100; Santa Cruz), rabbit anti-Olig1 (1:200; Millipore), rabbit anti-OTX2 (1:4,000; Millipore), mouse anti-PSA-NCAM (1:200; Santa Cruz), goat anti-SOX2 (1:200; R&D), mouse anti-synaptophysin (hSYP, 1:1,000; Enzo Life Sciences), sheep anti-TH (1:800; Pelfreeze), rabbit anti-TH (1:1,000; Pelfreeze). For quantification of HNA⁺, Ki67⁺, GFP⁺, and TH⁺ cells, images were captured at 20× magnification using a Zeiss Axio Observer Z.1 epifluorescence microscope. The density of Nestin labeling (percentage of immunoreactive pixels) was assessed from captured images and analyzed using ImageJ software.