Mouse anti nkx2

Lipopolysaccharide (LPS; Escherichia coli 0111:B4) was obtained from InvivoGen. Creatine monohydrate (C3630), 3-guanidinopropionoic acid (GPA; G6878), and carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP; 370–86-5) were obtained from Sigma-Aldrich. MitoTracker Red FM (M22425), tetramethylrhodamine, ethyl ester (TMRE; T669), and propidium iodide (PI) (P3566) were obtained from Thermo Fisher Scientific. Primary antibodies for immunohistochemistry (IHC) were as follows: rat anti-CD11b (1:100; AbD Serotec), rabbit anti-cleaved caspase-3 (1:100; Cell Signaling Technology), rabbit anti-Olig2 (1:300; Millipore), rat anti-myelin basic protein (MBP, 1:200; AbD Serotec), mouse anti-CC1 (1:300; Millipore), and mouse anti-Nkx2.2 (1:100; Developmental Studies Hybridoma Bank). Primary antibodies for immunocytochemistry (ICC) were as follows: rabbit anti-Olig2 (1:500; Millipore), rat anti-MBP (1:500; AbD Serotec), anti-GFAP (1:500; Sigma-Aldrich), mouse anti-CC1 (1:200; Millipore), and anti-PDGFRα (1:300; BD PharMingen). Alexa Fluor 488 or 594 secondary antibodies (Thermo Fisher Scientific) were used at a concentration of 1:500. To label nuclei, cell and tissues were labeled with 1 μg/ml Hoechst in PBS for 5 min at room temperature (RT) (33342; Thermo Fisher Scientific).

Different two-step protocols for bright field or immunohistofluorescence (IHF) were conducted after ISH. Antibody cocktails were used as follows: (1) Incubation at 4°C for 72 h in a mixture of mouse anti-Pax7, mouse anti-Nkx2.2 or mouse anti-Isl1 (Developmental Studies Hybridoma Bank, Iowa City, IA, USA; diluted 1:500 in PB containing 0.5−1% Triton X-100 [PB-T]) (Table 3) after hybridization; (2) second incubation was for 90 min in biotinylated horse anti-mouse (1:100, Vector, Burlingame, CA, USA; catalog reference: BA-2000); and (3) the avidin-biotin complex (ABC) standard kit (Vector, Burlingame, CA, USA: SK4100) was used next as recommended by the manufacturer, with appropriate washing steps. In the case of immunohistofluorescence, second incubation was for 90 min in Alexa 488-conjugated goat anti-mouse (Molecular Probes; catalog reference A21042), followed by washing steps to finish the reaction. In some cases, single bright field IHC was performed for Nkx2.2 or Pax7 as above, without combination with ISH.