Bungarotoxin staining

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1 total messages Started by draldua Tue, 30 Oct 2012 13:40

#47

Author: draldua
Date: Tue, 30 Oct 2012 13:40

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I have tried to label the NMJs in 48 hpf zebrafish embryos, by using Alexa Fluor 555-alpha bungarotoxin as a marker for post-synaptic structures and znp1 as PMNs marker. Following the protocol described by Wood et al. (2006)* I incubate the embryos for 30 min (rt) with the bungarotoxin (10 ug/mL), just after the 2 h incubation in the secondary antibody. Then I wash them 6x30 min in blocking buffer. The labelling of the PMNs is perfect, but for any unknown reason I have no labelling of the AChR. I have tried with two different batches of bungarotoxin, with and without bleaching of the embryos, with and without the acetone step, but I have not been able to overcome the problem. Any suggestion would be welcome! 

*Hum Mol Genet. 2006 Sep 15;15(18):2763-71. 
The microtubule-severing protein Spastin is essential for axon outgrowth in the zebrafish embryo.
Wood JD, Landers JA, Bingley M, McDermott CJ, Thomas-McArthur V, Gleadall LJ, Shaw PJ, Cunliffe VT.

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