Calbindin d28k

The specificity of the immunohistochemical reactions was assessed by replacing the primary antibody with TBS or the respective host serum as negative controls or by pre-absorbing the antibody against Calbindin D28k, ChAT, PGP 9.5 and Synaptophysin with its respective antigen (Calbindin D28k: Synaptic Systems, Göttingen, Germany; ChAT: Millipore, Billerica, MA, USA; PGP 9.5: Fitzgerald Ind., Acton, MA, USA; Synaptophysin: Synaptic Systems, Göttingen, Germany). For specificity control of GFAP, PLP, and MBP, we used cryosections of the cerebellum as positive controls as described above. Specificity of DsRed and GFP antibodies was confirmed by combined cell-type specific expression of PLP (red fluorescence in transgenic PLP-CreERT2 x tdT mice) and GFAP (green fluorescence in transgenic GFAP-EGFP x PLP-DsRed1 mice).

Freshly dissected cerebella from Mwk mice and wild-type littermates were fixed overnight in 4% paraformaldehyde, cryoprotected in 30% sucrose, embedded in OCT compound (TissueTek) and frozen. 10-μm sagittal sections were incubated for 30 min in 0.1 M glycine buffer (pH7.4), blocked for 1 h (10% goat serum, 0.3% Triton-X100 in PBS) and incubated with primary antibodies in block solution overnight at 4°C. The following antibodies were used: VGLUT1 (Synaptic Systems, 1:500), VGLUT2 (Synaptic Systems; 1:200), p-CaMKIV (Thr196) (Santa Cruz; 1:50) and Calbindin D28k (Synaptic Systems; 1:200). Sections were incubated with Alexa Fluor-labeled secondary antibodies (Invitrogen; 1:2000) for 3 h at room temperature before being mounted using DAPI-containing Vectashield medium (Vector Labs).