Vglut1

Free-floating brain sections (40-µm-thick) were washed with PBS and blocked by incubating with PBST containing 10% goat serum, 2% BSA and 0.5% Triton X-100 for 4 h at room temperature. Slices were then incubated with primary antibodies against PSD95 (ab12093, 1:250; Abcam), Vglut1 (135304; Synaptic Systems), Iba-1 (019-19741, 1:1000; Wako), or CD68 (MCA1957, 1:500; Bio-Rad, Hercules, CA, USA) in blocking solution for 48 h at 4 °C. Thereafter, brain slices were incubated with Alexa 488-conjugated anti-guinea pig, Alexa 448-conjugated anti-rabbit or Alexa 647-conjugated anti-goat secondary antibodies, as appropriate, or stained with rhodamine Red X (1:1000), for 4 h at room temperature, then washed with PBST, incubated with DAPI (1:5000) for 10 min, and slide-mounted with Prolong Gold Antifade (p36930; Invitrogen). Engulfed content was analyzed using IMARIS software; image processing and quantification of engulfed volume was performed as described previously [39 ]. For Sholl analyses, microglia intersections were quantified using a Sholl Analysis plugin, as described elsewhere [40 (link)].

The following primary antibodies were used: rat anti-CTIP2 1:500 (Abcam ab18465), rabbit anti-CUX-1 1:500 (Abcam, ab140042), rabbit anti-IBA-1 primary antibody (Abcam, ab178846), rabbit anti-IBA-1 primary antibody 1:500 (Wako, 4987481428584), rabbit anti-Caspr2 1:1000 (Abcam ab137052), rabbit MAPK1/ERK antibodies 1:1000 (Cell Signalling mAb#4695), rabbit anti-DSred primary antibody 1:500 (Takara, 632496) which binds Td-tomato protein, and guinea pig anti-VGLUT-1 1:1000 (Synaptic Systems 135 304).
The following secondary antibodies were used: goat anti-rabbit Alexa-488 secondary antibody 1:1000 (Abcam, ab150089), donkey anti-rabbit Alexa-488 1:1000 (Abcam ab150073), goat anti-guinea pig Alexa-647 secondary antibody 1:1000 (Abcam ab150187), donkey anti-rabbit Alexa-647 1:1000 (ABCAM ab150075), goat anti-rat Alexa-488 1:1000, (Abcam ab150157).

Sagittal brainstem sections including TBs of 80 μm thickness were taken from within 240 μm of the midline using a vibrating microtome (Leica, VT1200S). The tracer-injected brains were sliced coronally at 80 μm thickness. After 3 × 10 min washes in PBS, sections were transferred to a blocking solution containing 1% bovine serum albumin, 1% Triton X100, and 0.1% saponin in PBS. Tissue was incubated for 48 h at 4°C with the following primary antibodies diluted 1:100 in blocking solution: mouse monoclonal IgG1 anti-neurofilament (NF)-associated antigen, (3A10, Developmental Studies, Hybridoma Bank) and rat monoclonal IgG2a anti-myelin basic protein (MBP), (ab7349, Abcam). Coronal sections were additionally stained with antibodies directed against vesicular glutamate transporter 1 (VGLUT1; Synaptic Systems, 1:2000) to identify the calyx synapses in the MNTB. Tissue was then washed 3 × 10 min in PBS at room temperature, before incubation for 24 h at 4°C with secondary antibodies diluted 1:200 in blocking solution: Cy3 donkey anti-mouse, AlexaFluor 488 donkey anti-rat (715-545-153 and 715-166-151, respectively, Dianova) sections were rinsed 3 × 10 min in PBS, and coverslipped with Vectashield mounting medium.

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).

Mouse brains were freshly dissected and fixed in 4% PFA overnight at 4°C. The frozen tissue sections were prepared as previously described (91 (link)). Primary cultured neurons on a Lab-Tek chamber slides were fixed in 4% PFA for 30 minutes at 37°C. After blocking (5% goat or donkey serum [Sigma-Aldrich], 0.3% Triton X-100 in PBS [pH 7.4]), brain slices or cultured neurons were incubated with primary antibodies against GHSR1a (Santa Cruz Biotechnology, sc-10359, 1:100), DRD1 (Abcam, ab81296, 1:200), PSD95 (Cell Signaling Technology [CST], 3450, 1:400), VGLUT1 (Synaptic Systems, 135304, 1:400), MAP2 (Sigma-Aldrich, M4403, 1:300), DCX (Santa Cruz Biotechnology, sc271390, 1:100), and c-Fos (Synaptic Systems, 226308, 1:400) in mixture or separately. After washing with PBS, the slices or neurons were probed with appropriate cross-adsorbed secondary antibodies conjugated to Alexa Fluor 488, Alexa Fluor 594, or Alexa Fluor 647 (Thermo Fisher Scientific, 1:500). Images were collected on a Nikon Ti2 confocal microscope. Mean intensity or volume of different staining were analyzed using Nikon-Elements Advanced Research software accordingly.

Cells were fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X-100/PBS and blocked with 5% skim milk in TBS-Tween20 (TBS-T) buffer. All antibodies were diluted in 5% skim milk/TBS-T. For neuronal markers analysis, following antibodies were used: mouse anti-MAP2 (Sigma; 1:2,000 dilution), mouse anti-Neurofilament H (Smi-32; 1:2,000), mouse anti-neuronal nuclei (NeuN; 1:500), rabbit anti-Synapsin 1 (Syn1; 1:500), rabbit anti-MAP2 (all Millipore; 1:2,000), mouse anti-Tuj1 (Cell Signaling, 1:2,000), rabbit anti-postsynaptic density protein 95 (PSD95; 1:250), rabbit anti-Drebrin (both Abcam; 1:500), rabbit anti-pan voltage-gated Na2 + channels (pan Nav; Alomone; 1:250), anti-vesicular glutamate transporter 1 (vGlut1; Synaptic Systems; 1:500), and goat polyclonal anti-PTB1 (Abcam; 1:200). Rabbit antibodies against c9RANT products, anti-poly(GP), anti-poly(GA), anti-poly(GR), anti-poly(PA), and anti-poly(PR), were generated as described previously (Gendron et al., 2013 (link); Mann et al., 2013 (link)) and used at 1:1,000 dilutions. Secondary fluorescent antibodies (Invitrogen) were used at 1:1000 dilutions. Nuclei were stained with Hoechst 33258 (Invitrogen). Confocal microscopy was performed using Zeiss LSM 510 microscope.