555 fbtx

EDL muscles from Thy1-YFP16 mice expressing YFP in nerve endings were used to visualize NMJs. Following perfusion, muscles were dissected and incubated with Alexa Fluor 555-conjugated α-bungarotoxin (555-fBTX, Invitrogen, #B35451, 1:1,000 in 1× PBS) for 1 h. Muscles were then washed three times with 1× PBS and whole mounted in VECTASHIELD (Vector Laboratories). For Lynx1 IHC, EDL muscles were incubated for 1 h at room temperature in blocking buffer (1× PBS, 5% bovine serum albumin, 3% goat serum, 0.5% Triton-X), incubated overnight at 4°C in Lynx1 antibody diluted 1:10 in blocking buffer, washed three times with 1× PBS, incubated for 2 h at room temperature in Alexa Fluor 488-conjugated polyclonal anti-mouse IgG antibody (Invitrogen # A-11001, 1:1,000) and 555-fBTX (1:1,000) diluted in blocking buffer, washed three times with 1× PBS, and whole mounted in VECTASHIELD. NMJs were imaged using a Zeiss LSM 700 confocal microscope. Maximum intensity projections from confocal z-stacks were created using Zen Black software (Zeiss).