Sections were treated with either p‐JAK2 antibody, p‐STAT3 antibody, cyclin D1 antibody (Cell Signaling Technology), CDK6 antibody (Cell Signaling Technology) with neuron‐specific nuclear protein (NeuN) antibody (Chemicon), glial fibrillary acidic protein (GFAP) antibody (Santa), or ionized calcium‐binding adapter molecule‐1 (Iba‐1) antibody (Cell Signaling Technology); either cyclin D1 antibody or CDK6 antibody with p‐STAT3 antibody at 4°C overnight. The sections were then incubated with a secondary antibody (Invitrogen) for 1 h at 22 ± 2°C. The nuclei were stained with DAPI.
For double staining of NeuN, GFAP, Iba‐1, cyclin D1, or CDK6 and TUNEL, slices were incubated with the primary antibodies against NeuN, GFAP, Iba‐1, cyclin D1, or CDK6, respectively, at 4°C overnight, with PBS used as a negative control. Following three washes in PBS, the sections were incubated with fluorescent‐labeled secondary antibodies. Sections were then stained using the TUNEL assay kit according to the manufacturer's instructions and counterstained with DAPI.
For double staining of NeuN, GFAP, Iba‐1, cyclin D1, or CDK6 and TUNEL, slices were incubated with the primary antibodies against NeuN, GFAP, Iba‐1, cyclin D1, or CDK6, respectively, at 4°C overnight, with PBS used as a negative control. Following three washes in PBS, the sections were incubated with fluorescent‐labeled secondary antibodies. Sections were then stained using the TUNEL assay kit according to the manufacturer's instructions and counterstained with DAPI.