Ab5032

Cells were double labeled with 10 μg/ml of rabbit polyclonal anti-human NCAM antibody (AB5032; Millipore) followed by AlexaFluor 647-labeled goat anti-rabbit secondary antibody (Molecular Probes, Invitrogen, Eugene, OR, USA) and inactive endosialidase-GFP fusion protein [33 (link)] in PBS. Parallel samples were labeled with AlexaFluor 647 mouse anti-human alkaline phosphatase antibody (B4-78; BD Biosciences, San Jose, CA, USA) and FITC mouse anti-human CD44 antibody (BD Biosciences) according to the manufacturer’s instructions. Appropriate fluorescence minus one (FMO) controls were used for analysis. The cells were analyzed with FACS LSR II flow cytometer and FACSDiva 5.0.3 software (BD Biosciences). Cell debris and dead cells were excluded from the analysis based on physical parameters and propidium iodide (PI) fluorescence probing for cell viability (proportion of positive cells 1.2–7.0 %, data not shown).

Genioglossus and styloglossus muscles were embedded in OCT and frozen in isopentane pre-cooled in liquid nitrogen. For myofiber size analyses, thin sections (10 μm) were fixed on slides for 10 minutes in 4% paraformaldehyde (PFA), rinsed, blocked, and incubated overnight at 4°C in primary antibodies for Laminin gamma-1 (D18; DSHB, applied at 1:100) and Neural Cell Adhesion Molecule (NCAM) (Millipore, AB5032, applied at 1:250) [32 (link)]. After rinses and application of the secondary antibodies AF488 (1:800) and AF594 (1:500) for 1 hour at room temperature, slides were rinsed, mounted with DAPI, and imaged. For each staining iteration, negative control slides were prepared with primary antibodies omitted. Biological positive controls for muscle pathology consisted of concurrent staining of muscle tissue sectioned from a rat model of amyotrophic lateral sclerosis.

Immunohistochemistry using a rabbit anti-neural cell adhesion molecule (NCAM) antibody (AB5032, Millipore, Burlington, MA) and rabbit anti-Gαo antibody (551, MBL, Tokyo, Japan) was performed using olfactory organ sections from lungfish as described previously [23 (link), 28 (link)]. Sections were incubated with each primary antibody overnight at 4°C, washed, and then incubated with a secondary antibody, Alexa Fluor 488-donkey anti-rabbit IgG (A21208, Thermo Fisher Scientific, Waltham, MA) for 2 h at RT. The sections were mounted in VectaShield mounting medium with DAPI (H-1200, Vector Laboratories, Burlingame, CA).

For immunocytochemical staining, cells were grown on glass coverslips and fixed with 4 % paraformaldehyde in PBS. Nonspecific binding was blocked with 1.5 % normal horse serum (Vector Laboratories, Burlingame, CA, USA) in PBS. Cells were labeled with 10 μg/ml of rabbit polyclonal anti-human NCAM antibody (AB5032; Millipore) followed by AlexaFluor 647 conjugated goat anti-rabbit secondary antibody (Molecular Probes) and inactive endosialidase-GFP fusion protein [33 (link)], all in PBS. Cover slips were mounted with ProLong Mounting Medium with DAPI (Molecular Probes). The staining was visualized with an Olympus BX50F-3 microscope and imaged by a PCO CCD Imaging SensiCam color camera and Image-Pro Plus 4.0 software.

30-μm brain sections were washed in PBS and blocked for 1 hour in 5% normal donkey serum (NDS, Jackson Immunoresearch) and 0.3% Triton X-100 (Sigma-Aldrich) in PBS. Incubation with the primary antibodies was done overnight at 4°C in blocking solution. After 3 × 10 minutes washes with PBS, the sections were incubated with the corresponding secondary donkey Alexa fluorescent antibodies (1:1,000, Thermo Fisher) and 0.1 mg/ml of 40-6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich) for 2 hours at room temperature (RT). Sections were finally washed with PBS 3 × 10 minutes, mounted on glass slides in a 0.2% solution of gelatin (Sigma-Aldrich) in 0.05 M Tris-HCl buffer (pH 7.4), dried and cover slipped in Dako fluorescent mounting medium. The primary antibodies used in this study were the following: Goat anti GFRα1 (1:500, AF560, RnD); Goat anti ChAT (1:500, AP144P, Millipore); Chicken anti GFP (1:500, ab13970, Abcam); rabbit anti NCAM (1:1,000, AB5032, Millipore); rabbit anti cFOS (1:500, sc-52, Santa Cruz, USA); rabbit anti calretinin (1:500, AB5054, Chemicon, USA); mouse anti calretinin (1:500, 6B3, Swant, Switzerland); rabbit anti GluA1 (1:500, AB1504, Millipore); rabbit anti GluA2 (1:500, AB1768-I, Millipore); rabbit anti GluA3 (1:500, AGC-010, Alomone Labs); rabbit anti GluA4 (1:500, AB1508, Millipore); and rat anti SP (1:500, Millipore, MAB356).

Primary antibodies used were: NCAM-1 (1:100, rabbit, Millipore, ab5032; rat, Millipore, mab310); ALCAM (1:50, goat, R&D Systems, AF1172); CNTN2 (1:50, goat, R&D Systems, AF4439); Cx40 (1:500, rabbit, Alpha Diagnostic, Cx40A); PSA (1:500, rabbit, Absolute Antibody, Ab00240-23.0); troponin T (1:100, mouse, Thermo Fisher Scientific MS-295-P0); plakoglobin (1:100, rabbit, Cell Signaling Technology, 2309S). Secondary antibodies used were: donkey anti-rabbit 555 (1:500, Thermo Fisher Scientific, A31572); donkey anti-goat 555 (1:500, Thermo Fisher Scientific, A21432); goat anti-rat 555 (1:500, Thermo Fisher Scientific, A21434); goat anti-rabbit 488 (1:500, Thermo Fisher Scientific, A11034); donkey anti-rabbit 488 (1:500, Thermo Fisher Scientific, A21206); donkey anti-goat 488 (1:500, Thermo Fisher Scientific, A11055); donkey anti-mouse 488 (1:500, Thermo Fisher Scientific, A21202); donkey anti-rat 488 (1:500, Thermo Fisher Scientific, A21208); wheat germ agglutinin (WGA) Alexa Fluor 488 conjugate (1:250, Thermo Fisher Scientific, W11261).