Rabbit anti pkcγ

The whole cerebellum from PKCγ-S361G transgenic mice was homogenized in ice-cold RIPA buffer with added protease- and phosphatase-inhibitors (Roche, Mannheim, Germany) (50 mM Tris-HCl, pH 7.4; 0.15 M NaCl, 0.25% deoxycholic acid/sodium deoxycholate, 1% NP-40, 1 mM EDTA). Samples were homogenized using an ultrasound probe and then centrifuged at 14500g for 15 min. Immunoprecipitations were performed using the Pierce® Crosslink Immunoprecipitation Kit (Thermo Fisher Scientific, Rockford, USA) according to standard protocols. Briefly, 5 μg of primary rabbit anti-CRMP2 (Sigma-Aldrich, St. Louis, USA; C2993), rabbit anti-PKCγ (Santa Cruz Biotechnology, Santa Cruz, USA; sc-211) or normal rabbit IgG (Santa Cruz, Santa Cruz, USA; sc-2027) was incubated with protein A/G-agarose beads for 1 h at room temperature. The antibodies were crosslinked to the beads for 30 min using disuccinimidyl suberate. Lysates were precleared with control agarose resin for 1 h at 4 °C. Seven hundred fifty micrograms of protein in 400 μL immunoprecipitation buffer was incubated with each type of antibody-bead overnight at 4 °C on a rotating platform. Beads were washed 6x with IP buffer and proteins were eluted under acidic conditions. The pH was neutralized with 1 M Tris buffer. Samples were mixed in 2x Laemmli buffer (Sigma-Aldrich, Buchs, Switzerland) and processed via SDS-PAGE and Western blotting.

Mice were deeply anesthetized with ketamine/xylazine cocktail, transcardially perfused with 4% paraformaldehyde in phosphate buffered saline. Spinal cord and brain were dissected and cryopreserved in 30% sucrose and cryosectioned. Free floating sections (40 µm) were incubated in 0.3% bovine serum albumin in 0.3% Triton X-100 for 30 min, then incubated overnight at 4 °C with primary antibody. The next day, sections were washed and incubated with Alexa Fluor conjugated secondary antibody (1:250; Invitrogen) for 3 h at room temperature then, washed and, sections cover-slipped in Fluoroshield with DAPI (Millipore). Antibodies used for fluorescent immunohistochemistry were: Rabbit anti-PKCγ (1:100; Santa Cruz) and rabbit anti RFP (1:100; Rockland). PKCγ immunoreactivity was used to assess the completeness of the corticospinal transection at the medullary pyramids. RFP was used to label corticospinal neurons in the optogenetic inhibition experiment.

All the immunohistochemistry experiments were performed on at least 3 animals per age and per genotype. For light microscopy, P0-P2 mice were anesthetized on ice and adult mice were anesthetized with sodium pentobarbital (50 mg/kg i.p.). Embryos were fixed by immersion in 4% paraformaldehyde with 0.12 M phosphate buffer pH 7.4, and post-natal mice were perfused through the aorta with 0.12 M phosphate buffer, pH 7.4, containing 4% paraformaldehyde. Tissue preparation and immunostaining were carried out as described in ref. 59 (link), using the following primary antibodies: goat anti-DCC (1/100; Santa Cruz Biotechnology, Santa Cruz, California); rabbit anti-PKCγ (1/100, Santa Cruz) to reveal the CST in mature mice after P2; chicken anti-GFP (1/500, Aveslab) to reveal the CST in both newborn and mature Emx1::cre;Tau^mGFP mice ; mouse anti-MBP (1/200, Chemicon, Millipore, Molsheim, France) to reveal corpus callosum in adult mice and rat anti-L1 (1/400, Millipore) to reveal corpus callosum before myelination.

After recording, spinal cord slices containing LY-injected neurons were fixed for 30 min with 4% paraformaldehyde in phosphate buffer (PB; 0.1 M, pH 7.4). After several washings in phosphate buffered saline (PBS; 0.05 M, pH 7.4), slices were pre-incubated in PBS with 1% normal goat serum for 30 min and then incubated overnight at 4 °C in rabbit anti-NK1 1:10000 (Sigma) for LI neurons, rabbit anti-PKCγ (1:100, Santa Cruz Biotechnology) or biotinylated isolectin B4 (1:1000, Sigma) for LII neurons. Slices were then washed in PBS and incubated for 3 h with anti-rabbit AlexaFluor 495 1:500 (Molecular Probes, Carlsbad, CA) or 1 h with Extravidin-Cy3 1:1000 (Sigma). Slices were finally mounted in Vectashield H-1000 mounting medium (Vector Lab). Confocal laser scanning microscopy was performed using a Zeiss LSM 510 confocal microscope with a 63x oil lens.