Phosphate buffer: 0.1 M phosphate buffer (PB, pH =7.4), 0.01 M phosphate buffer saline (PBS, pH = 7.4)
Normal saline (0.9% NaCl)
Paraformaldehyde (PFA; Shanghai Sangon)
Triton X-100 (Sigma)
Embedding medium for frozen sectioning, O.C.T. compounds (Sakura)
Normal serum of goat, the host species of the secondary antibodies (Invitrogen)
Mounting medium for fluorescent sections (H-1000, Vector)
Nail varnish (use those that dry quickly in a few seconds)
Salts for artificial cerebrospinal fluid (ACSF) and sucrose solution (Amresco)
Super glue (Pattex)
1) anti-Nav1.2 IgG (ASC-002, Alomone), use 1:200 dilution
2) anti-Nav1.6 IgG (ASC-009, Alomone), use 1:200 dilution
3) anti-Pan-Nav IgG (ASC-003, Alomone), use 1:200 dilution
4) anti-AnkG IgG (SC-12719, Santa Cruz), use 1:200 dilution
1) Anti-rabbit IgG: Alexa Fluor 488 goat anti-rabbit IgG (A31627, Invitrogen), use 1:1,000 dilution
2) Anti-mouse IgG: Alexa Fluor 594 goat anti-mouse IgG (A31624, Invitrogen), use 1:1,000 dilution
Fixative Dissolve 1 g PFA in 100 ml 0.1 M PB in 60 °C water bath. Allow to cool at room temperature, add 1 g sucrose and then filter. This solution can be stored at 4 °C for several days.
5% Triton X-100 stock solution Add 5 ml of Triton X-100 into 95 ml of 0.01 M PBS, stir gently until dissolved, and store at 4 °C.
0.3% Triton X-100 Add 3 ml of 5% Triton X-100 into 47 ml PBS, stir gently until dissolved, and store at 4 °C.
0.1% Triton X-100 Add 15 ml of 0.3% Triton X-100 into 30 ml PBS, stir gently until dissolved, and store at 4 °C.
Blocking and permeabilizing solution 5% normal goat serum in 0.3% Triton X-100. CRITICAL Make up fresh before use.
Antibody dilution solution 5% normal goat serum in 0.1% Triton X-100. CRITICAL Make up fresh before use.
ACSF containing (in mM) NaCl 126, KCl 2.5, MgSO4 2, CaCl2, 2, NaHCO3 26, NaH2PO4 1.25, dextrose 25 (315 mOsm, pH 7.4)
Ice-cold aerated slicing solution (sucrose solution) sucrose-substituted ACSF in which equiosmolar sucrose is used as a substitute for NaCl, containing (in mM) KCl 2.5, MgSO4 2, CaCl2 2, NaHCO3 26, NaH2PO4 1.25, dextrose 10, sucrose 213.
Cryoprotectant Dissolve 30 g sucrose in 0.1 M PB to a final volume of 100 ml, and store at 4 °C.