Alexafluor 647 antibody

Samples were fixed for 35 min in 4% paraformaldehyde (EMS, cat. no. 15710), washed three times with 1 × PBS, and permeabilized in 0.3% Triton-X100 + 4% normal horse serum (NHS) (Sigma) for 60 min. Samples were blocked in 4% NHS (Sigma), and all subsequent steps were performed using 4% NHS for antibody dilutions. For staining of actin and acetylcholine receptors, samples were incubated with AlexFluor-488-conjugated phalloidin (1:200, Invitrogen, A12379) and AlexaFluor-647-conjugated bungarotoxin (1:250, Invitrogen, B35450) respectively. For morphological assessment of motor neurons and endothelial cells, fixed samples were incubated with an axonal marker SMI-35 (1:250, Abcam, ab18207) or motor neuron-specific marker for choline acetyltransferase (ChAT) (1:200, Abcam, ab18736), presynaptic marker synaptophysin (1:500, Abcam, ab32127), and endothelial surface marker CD31 (1:500, Abcam, ab32127) for 16 h at 4 °C followed by AlexaFluor-568 antibody and AlexaFluor-647 antibody (Life Technologies). Images were acquired using a Nikon Eclipse TI A1RSI laser scanning confocal microscope.

Samples were fixed for 15 min in 4% paraformaldehyde (Alfa Aesar), washed three times with 1× PBS, and permeabilized in 0.5% Triton-X100 (Sigma) for 15 min. Samples were blocked in 1% bovine serum albumin (Sigma) and all subsequent steps were performed using 0.1% bovine serum albumin for antibody dilutions. For staining of actin, samples were incubated with Alexfluor-488-conjugated phalloidin (1:100, Life Technologies). For assessment of myotube morphology and maturity, fixed samples were incubated with a fast MHC marker (Abcam) for 16 h at 4 °C followed by Alexa Fluor-647 antibody (Life Technologies). For co-staining of endothelial cells, samples were then incubated with CD31 antibody (Dako) for 16 h at 4 °C followed by Alexa Fluor-594 antibody (Life Technologies). Images were acquired using a Zeiss LSM710 confocal microscope.