Alexa fluor 555 goat anti chicken

At day 14 in vitro, the cultures were fixed with 4% paraformaldehyde for 10 min, rinsed 3× with HEPES Buffered Hank’s saline (HBHS) (in g/L; 7.5 g NaCl, 0.3 g KCl, 0.06 g KH2PO4, 0.13 g Na2HPO4, 2 g Glucose, 2.4 g HEPES, 0.05 g MgCl2:6H2O, 0.05 g MgSO4:7H2O, 0.165 g CaCl2, 90 mg NaN3, at pH 7.4), then permeabilized with 0.2% Triton-X (Sigma-Aldrich, St. Louis, MO). The cultures were then blocked with 10% normal goat serum (Jackson Immunoresearch, West Grove, PA) for 1 h, after which primary antibodies to beta-3-tubulin (β-3-tub) (Covance, Princeton, NJ), which labels neurons; Glial Fibrillary Acidic Protein (GFAP) (Millipore, Billerica, MA), which labels astrocytes; and Ionized Calcium binding adaptor molecule 1 (Iba1) (Wako, Osaka, Japan), which labels microglia, were added, and the cultures incubated in a 4°C refrigerator overnight. The wells were then aspirated, rinsed in HBHS 3×, and the following secondary antibodies were added: Alexa Fluor 488 Goat anti-mouse, Alexa Fluor 555 Goat anti-chicken, and Alexa Fluor 635 Goat anti-rabbit (Invitrogen, Carlsbad, CA). After a 2 h incubation at room temperature, the secondary antibodies were rinsed 3× with HBHS, and a final volume of 100 μl of HBHS was left in the wells for imaging. Special care was taken to ensure the microwire segments remained attached to the bottom of the wells.

The round microscope coverslips with Neuro2a were removed from 6-well plates to be fixe for 60 min in a paraformaldehyde (PFA) solution and immediately used for immunocytochemistry. The fixed cells were incubated overnight at 4°C in PBS 1X containing 5% native goat serum, 1% BSA with rabbit anti-Na_X antibody targeting the interdomain 2–3 region of the Na_X channel's α-subunit (1/250) and mouse anti-NeuN (1/500, Clone A60, Millipore) or mouse anti-Na⁺/K⁺-ATPase α3 subunit (1/10, Clone XVIF9-G10, Sigma-Aldrich) or mouse anti-Na⁺/K⁺-ATPase α1 subunit (1/10, Clone C464.6, Millipore) or chicken anti-MAP-2 antibody (1/100, Polyclonal Antibody, Millipore). The cells were first washed in PBS and incubated for 2 h at room temperature in PBS 1Xcontaining AlexaFluor-488 goat anti-rabbit (1/500, green, Invitrogen), and AlexaFluor-555 goat anti-mouse (1/500, red, Invitrogen) or AlexaFluor-555 goat anti-chicken (1/500, red, Invitrogen) as secondary fluorescent antibodies to visualize the Na_X and NeuN or Na⁺/K⁺-ATPase α3 or α1 isoforms or MAP-2 proteins, respectively. The specificity of the Na_X antibody has been tested using SCN7A control peptide (Cedarlane; 68912-12) at different concentration from 0 to 10 μg in differentiated Neuro2a cells (see Supplemental Figure 1).

Slices were washed in phosphate-buffered saline (PBS) three times for 15 min and then blocked for 1 h in 0.5 mL of blocking solution (100 mM PBS, 0.005% bovine serum albumin, 0.4% Triton X-100, and 10% donkey serum). Slices were washed again and immersed in the primary antibody solution (10 mM PBS, 1% donkey serum, 0.1% sodium azide, 0.4% Triton X-100, and primary antibody) for 48 h at room temperature with continuous agitation. After a third washing step, slices were immersed in the secondary antibody solution (10 mM PBS, 1% serum, 0.1% sodium azide, 0.4% Triton X-100, and secondary antibody) and agitated continuously for 24 h. Slices were washed a final time with PBS, mounted onto 0.1% gelatin-subbed slides, embedded in DAPI-Fluoromount (Southern Biotech), and coverslipped. Details of the primary antibodies used are as follows: anti-GFP, chicken polyclonal (Aves, Davis, CA, USA, RRID:AB_10000240, 1:1000); anti-RGS14 monoclonal antibody (Neuromab, Davis, CA, USA, RRID:AB_10698026). Details of the secondary antibodies used are as follows (all diluted 1:1000): goat anti-chicken Alexa Fluor^® 555 (ThermoFisher Scientific, Waltham, MA, USA, A-21437, RRID:AB_2535858); goat anti-mouse Alexa Fluor^® 568 (Thermo Fisher Scientific, #A-21134, RRID:AB_2535773).