Alexa fluorophore conjugated secondary antibody

Cerebellar slices were washed in PBS, permeabilized and blocked in 4% goat serum and 0.1% Triton X‐100 in PBS (blocking buffer) for 1 h and incubated overnight at 4°C with primary antibodies (Table 2). Slices were washed in PBS with 0.1% Triton X‐100 and incubated with Alexa fluorophore‐conjugated secondary antibodies (1:400; Invitrogen) in blocking buffer for 1 h at RT. Slides with cryostat spinal cord sections were air‐dried for 1 h, rehydrated in Tris buffer saline (TBS; 20 mM Tris and 1.4 M NaCl in dH₂O; pH 7.6) and pre‐treated with absolute ethanol (Sharlab) for 15 min at −20°C. For APC and Olig2 immunostaining, antigen retrieval was performed by heating the sections in low‐pH retrieval buffer (Vector Laboratories) for 45 s using a microwave. After washing, samples were incubated in blocking buffer solution (1% BSA, 5% goat serum, and 0.1% Triton X‐100) for 30 min at RT, and then with the primary antibodies diluted in blocking buffer overnight at 4°C (Table 2). Sections were washed in TBS, and incubated with Alexa fluorophore‐conjugated secondary antibodies (1:500; Invitrogen) in blocking solution for 1 h at RT. Cell nuclei were counterstained with DAPI (4 μg/ml, Sigma‐Aldrich) and sections were mounted with Fluoromount‐G (SouthernBiotech).