Mouse anti calb calbindin

For immunofluorescence experiments, 40–50 μm floating sections were collected in 1× PBS, then rinsed once in 1× PBT (PBS + 1% Triton 100×), and incubated overnight at 4 °C in a cocktail of primary antibodies that were diluted in 1× PBT + 20% normal donkey serum. After the overnight incubation, sections were rinsed three times with 1× PBT for 10 min and incubated for 1.5 h in the corresponding secondary antibodies (1:500, Jackson-ImmunoResearch or Invitrogen). Cerebellar sections were then washed three times with 1× PBT for 10–15 min, incubated with DAPI, and mounted in Poly-aquamount (Polysciences). The following primary antibodies were used: rabbit anti-IBA1 (1:200, Wako, # 019-19741), mouse anti-CALB (calbindin, 1:200, Sigma-Aldrich, #C9848), rat anti-CD68 (1:200, Bio-Rad, #MCA1957), Lycopersicon esculentum(Tomato)-Lectin (1:200, Sigma-Aldrich, #L0401), guinea-pig anti-VGLUT1 (1:800, Synaptic Systems, #135404), rabbit anti-phosphorylated S6R (1:200, Cell Signaling, #2211), rabbit anti-phosphorylated AKT (1:200, Cell Signaling, #8112), rabbit anti-TFEB (1:200, Bethyl Laboratories, A303-673A), rabbit anti-FASN (1:200, Cell Signaling, #3180), rabbit anti-pyruvate dehydrogenase E 1 alpha (PDE) (1:200, GeneTex, # GTX104015), rat anti-CD107a (LAMP-1) (1:200, BioLegend, # 121602).

For immunostaining, brain sections in slides (30 μm) or as floating sections (50 μm) were rinsed once in 1X PBT (PBS + 1% Triton 100X) and incubated in primary antibodies diluted with 1X PBT + 20% normal donkey serum for two nights at 4 °C. After incubation with primary antibodies, sections were rinsed three times with 1X PBT for 10 min and incubated for two hours in the corresponding secondary antibodies (1:800, Jackson-ImmunoResearch). Tissue was then washed three times with 1X PBT for 10–15 min, incubated with DAPI and mounted in Poly aquamount (Polysciences). The following primary antibodies were used: rabbit anti-IBA1 (1:200, Wako), mouse anti-CALB (calbindin,1:200, Sigma-Aldrich), rat anti-CD68 (1:200, Biorad), and rat anti-CD206 (1:200, Bio-Rad). To stain PC somata, the NeuroTrace™ 530/615 Red Fluorescent Nissl Stain was used (NT, 1:80, ThermoFisher Scientific).