Macro h2a 1

Morphological studies were carried out on cresyl violet (Nissl), and hematoxylin and eosin stained paraffin sections, as described previously[18 (link)]. Immunohistochemical staining was performed on 3–5 μm paraffin sections as well as on 50–100 μm vibratome sections, both using fluorescence detection. Sections of the spinal cord were stained with antibodies to β-tubulin III (2 μg/mL), nestin (2 μg/mL; R&D), SOX2 (5 μg/mL; BD Biosciences), MAP2 (5 μg/mL; Sigma-Aldrich), NF200 (5 μg/mL; Sigma-Aldrich), GFAP (2 μg/mL; DAKO), brain derived neurotrophic factor (5 μg/mL; Abcam), and macro H2A.1 (Abcam). Secondary antibodies used were Alexa Fluor 488 goat anti-mouse IgG (H + L) and Alexa Fluor 633 goat anti-rabbit IgG (H + L) (highly cross-absorbed, all dilutions 1:400; Invitrogen, United States); counterstaining was done with Hoechst. Fluorescence was detected by a confocal microscope Nikon A1 (Nikon, Japan). For the quantification of positive cells, we used NIS Elements software (Nikon).

Cells were cultured for 14 d in complete growth medium followed by fixation with ice-cold buffered 4% paraformaldehyde for 30 min. For flow cytometry cells were detached with Stem Pro Accutase followed by fixation with ice-cold buffered 4% paraformaldehyde for 10 min. The following antibodies were used: nestin (R and D and Abcam), SOX2 (BD Biosciences), βIII-tubulin (R&D), microtubule associated protein 2 (MAP2) (Sigma-Aldrich), glial fibrillary acidic protein (GFAP) (DAKO), NF-200 (Sigma-Aldrich), macro H2A.1 (Abcam), human leukocyte antigen (HLA)-ABC (BD Pharmingen), and HLA-DR (Miltenyi Biotec). For flow cytometry, directly labeled primary antibodies were used at a concentration of 10 µg/mL. For ICC, all primary antibodies were diluted in PBS-TT (PBS with 0.2% Tween 20, 0.3% Triton X-100, and 1.0% normal goat serum) at a concentration of 1-5 µg/mL. Goat anti-mouse IgG (H + L) labeled with Alexa Fluor 488 and goat anti-rabbit IgG (H + L) labeled with Alexa Fluor 633 (Life Tech, United States), all diluted at 1:400 in PBS-TT, served as secondary antibodies. Cell nuclei were counterstained with Hoechst (1 µg/mL; Invitrogen, United States). A Nikon A1 scanning laser confocal microscope (Nikon Company, Japan) was used to evaluate all ICC, while an S3e cytometer (Bio-Rad) was used for flow cytometry.