Anti calbindin d28k

Protein samples from whole cell extracts or subcellular fractionation (ThermoFisher Scientific, Waltham, MA, USA) were isolated accordingly. Samples were loaded in SDS-PAGE after BCA protein quantification (Bio-Rad, Hercules, CA, USA). The protein was transferred from the gel onto nitrocellulose membranes (Bio-Rad) and incubated overnight at 4 °C with the following primary antibodies: anti-Calbindin D28K (Santa Cruz Biotechnology, Dallas, TX, USA, sc-7691, sc-365360; 1:400), anti-TFII-1 (Santa Cruz Biotechnology sc-28716, 1:500), and NFATc1 (Pierce MA3-024, Santa Cruz Biotechnology, 1:400; sc-7294). Fluorescent-labeled secondary antibodies (Li-Cor, Lincoln, NE, USA) were incubated for 1 h at room temperature followed by washes on an orbital shaker with TBS 0.05% Tween-20. Blots were then scanned with a Li-Cor Odyssey scanner (Li-Cor, Lincoln, NE, USA).

Immunofluorescence was performed as described by Dong et al. (2016 (link)). After deparaffinization and washing, the preincubated sections were incubated with the mouse monoclonal antibody anti-Calbindin-D-28 K (Sigma-Aldrich, St. Louis, MO, USA; ratio of 1:3000) and the rabbit polyclonal antibody anti-GAD65 (Santa Cruz Biotechnology, Inc., USA; ratio of 1:50), or the mouse monoclonal antibody anti-Calbindin-D-28 K and the rabbit polyclonal antibody anti-Neurofascin (Santa Cruz Biotechnology, Inc., USA; ratio of 1:25) for double immunofluorescence labeling. Then, tissue sections were washed in PBS after overnight at 4°C, and incubated for 2 h using secondary antibody conjugated to the fluorescent markers FITC and Rhodamine (Zhongshan Biotechnology, Beijing, China; ratio of 1:100). Finally, tissue sections were mounted with glycerin gelatin for histological examination. The slices labeled by FITC and Rhodamine were observed by a fluorescence microscope (BX61+DP-71; Olympus/IPP, Japan/USA). And these images were obtained from cerebellar lobule 4–5 and then merged at a magnification of × 400 (ocular × 10 and objective × 40), respectively. The mean intensities of GAD65 and NF186 in the cerebellum were obtained by using image analysis program (MetaMorph, UIC, US). Three different fields per section were selected, and three sections per animal were measured to get a mean value.