H 215

After perfusion, a laminectomy was performed and the lumbar enlargement of the spinal cord was removed. The lumbar cord was sectioned into 50 µm thick horizontal sections and the sections containing traced motoneurones were selected for immunohistochemistry. For AIS labelling an antibody against Ankyrin G (rabbit anti-AnkG, 1:500, H215, Santa Cruz Cat no: sc28561) was used and for C-bouton labelling an antibody against the vesicular acetylcholine transporter was used (goat anti-VAChT, 1:500, Millipore, lot no. 2778849, ABN100). Immunohistochemistry was performed on free-floating horizontal sections. The tissue was first incubated in a blocking solution (5% normal donkey serum in PBST (phosphate buffered saline with 0.3% triton X-100) for 4 hours followed by overnight incubation with the primary antibodies diluted in blocking solution. The tissue was then sequentially incubated in the dark for 2 hours for each secondary antibody - donkey anti-rabbit Alexa Fluor 568 (Thermo Fisher Scientific Cat no: A-11055, 1:1000) and donkey anti-goat DyLight 650 (Invitrogen, lot nr. SA2325947, 1:1000). Sections were then rinsed, mounted and cover-slipped with fluorescent mounting medium (Dako Denmark).

At the indicated time, neurons on coverslips were rinsed briefly with D-PBS and fixed by a sequential paraformaldehyde/methanol fixation procedure^{69 (link)}. The following antibodies were used for immunostaining: primary antibodies to tubulin α-subunit (mouse monoclonal 12G10, 1:1000 dilution; Developmental Studies Hybridoma Bank, University of Iowa, IO), ankyrin G (rabbit polyclonal H-215, 1:50 dilution; Santa Cruz Biotechnology Inc., CA), MAP2 (mouse monoclonal clone HM-2; 1:500; Sigma, MO), Tau (rabbit polyclonal LF-PA0172; 1:500; Abfrontier, Seoul, Korea), Hnrnpa2b1 (Goat polyclonal, 1:500; Santa Cruz Biotechnology Inc., CA), Map1b (rabbit polyclonal; 1:500; Cell Signaling Technology, Danvers, MA), and secondary antibodies (Alexa Fluor 488-conjugated goat anti-mouse IgG [1:1,000], Alexa Fluor 568-conjugated goat anti-rabbit IgG [1:1,000], Alexa Fluor 568-conjugated rabbit anti-goat IgG [1:1,000], Molecular Probes, OR). Neurons were then incubated with primary antibodies followed by secondary antibodies and mounted on slides as previously described⁷⁴ .

Hippocampal cultures were fixed via a sequential paraformaldehyde/methanol fixation procedure described previously [52 (link)]. Immunostaining was performed after blocking the hippocampal cultures, followed by the primary antibody treatment overnight at 4 °C. Primary antibodies used for immunostaining were as follows: primary antibodies to MAP2 (chicken polyclonal clone PA1-10005; 1:5000; Invitrogen, Waltham, MA, USA), tubulin α-subunit (mouse monoclonal 12G10, 1:1000 dilution; Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA, USA), ankyrin G (rabbit polyclonal H-215, 1:50 dilution; Santa Cruz Biotechnology Inc., Delaware Ave, CA, USA), tau (rabbit, polyclonal, LF-PA0172, 1:500, Abfrontier, Seoul, South Korea), and Glial fibrillary acidic protein (GFAP) (G-A-5; mouse monoclonal (G3893), 1:200, Sigma Aldrich, Saint Louis, MO, USA). After washing the primary antibody, Alexa-conjugated secondary antibody (Invitrogen) incubation was performed as indicated and mounted on slides. Imaging was performed using the Olympus BX53^® polarising light microscope (details in Section 4.10).