α bungarotoxin tritc

EDL muscles were fixed in 1% paraformaldehyde overnight. The muscles were washed in PBS and permeabilized in 2.5% Triton X-100 (Sigma-Aldrich) in PBS for 30 min and incubated in 1 µg/ml α-bungarotoxin-TRITC (Invitrogen) in 1 M NaCl for 30 min. Subsequently, muscles were incubated for 1 h in a blocking solution [4% bovine serum albumin (BSA), 0.5% Triton X-100]. After blocking, the muscles were incubated with a polyclonal chicken anti-neurofilament antibody (2BScientific) 1:500 in blocking solution overnight at 4°C, followed by incubation for 4 h with anti-chicken Alexa Fluor 488 antibody (Jackson ImmunoResearch). Finally, the muscles were mounted on slides with 1.8% low-melting point agarose (Thermo Fisher Scientific) and images were taken using a Zeiss LSM700 confocal microscope. The first author was blinded during image acquisition.

At 48 h post injection with 10 pg ATG poly-GR, 10 pg TAG poly-GR, or only 400 pg mCherry mRNA, wild-type AB zebrafish were fixed overnight in 4% paraformaldehyde. Fish were permeabilized with acetone for 1 h at −20°C and blocked with 1% BSA, 1% DMSO and PBS-T for 1 h at room temperature. Subsequently, fish were stained for 30 min with 1 µg/ml α-bungarotoxin-TRITC (Invitrogen), washed with PBS and incubated overnight at 4°C with the anti-mouse IgG SV2 antibody (1:200, AB231587, Developmental Studies Hybridoma Bank, University of Iowa). After incubation, fish were washed ten times in 1.5% PBS-T and incubated overnight with the secondary antibody anti-mouse-Cy5 (1:200, Sigma-Aldrich). The following day, the fish were washed six times in 1.5% PBS-T and mounted with 1.8% low-melting-point agarose (Thermo Fisher Scientific). Imaging was performed using a Leica SP5 AOBS confocal microscope and an HCX L 20.0×1.00 water dipping objective. N was ten fish per group. The intensity of the SV2 staining was measured for n=5 independent fish per group, across three neurites per fish, and corrected for background fluorescence. We performed a one-tailed unpaired t-test with Welch's correction.