Data on the specificity of an antibody to Drosophila vesicular acetylcholine transporter

The role of the vesicular acetylcholine transporter (VAChT) in the regulation of cholinergic neurotransmission has not been fully elucidated. Here we sought to develop a tool for studying vesicular acetylcholine transporter function, and we present data on the validation of our new anti-VAChT antibody. We show that the immunoreactivity of the antibody is not due to an artifact of secondary antibody staining, and we present two additional validation data. First, the peptide epitope used to generate the antibody is able to block the binding of the anti-VAChT antibody in vivo. Further, RNA interference (RNAi) -mediated knockdown of VAChT function in cholinergic neurons drastically reduces anti-VAChT staining in those constructs. Additional evidence for the antibody functionality is presented in our research article on the subject (Boppana et al., 2017) [1].


a b s t r a c t
The role of the vesicular acetylcholine transporter (VAChT) in the regulation of cholinergic neurotransmission has not been fully elucidated. Here we sought to develop a tool for studying vesicular acetylcholine transporter function, and we present data on the validation of our new anti-VAChT antibody. We show that the immunoreactivity of the antibody is not due to an artifact of secondary antibody staining, and we present two additional validation data. First, the peptide epitope used to generate the antibody is able to block the binding of the anti-VAChT antibody in vivo. Further, RNA interference (RNAi) -mediated knockdown of VAChT function in cholinergic neurons drastically reduces anti-VAChT staining in those constructs. Additional evidence for the antibody functionality is presented in our research article on the subject (

Experimental features
Samples were prepared using a protocol for whole-mount immunohistochemistry as described previously [2]. Briefly, Drosophila brains were dissected and immunostained as described elsewhere [1]. Imaging was then carried out using confocal microscopy as mentioned above.

Data source location N/A Data accessibility
The data are with this article

Value of the data
Describes a key resource for dissecting cholinergic neuron function in specialized neural circuits in the Drosophila nervous system.
The data may be relevant for current efforts to map the circuitry of neurotransmitter systems within the brain.
Describes two independent RNAi knockdown fly constructs of VAChT in cholinergic neurons that can be used to test the biological function of VAChT in those neurons in vivo.
The in vivo peptide titration experimental procedure could be useful in validating other antibody reagents besides those developed against vesicular transporters.

Data
The data described here shows the specificity of the Drosophila anti-VAChT antibody. In Drosophila whole mount immunohistochemical preparations, we present data indicating that the VAChT antibody staining is not due to a secondary antibody artifact. Moreover, we show a dramatic reduction in VAChT immunostaining in fly constructs with an RNAi knockdown of VAChT expression in cholinergic neurons. Importantly, we present data on the titration of the VAChT antibody against the peptide that was used to generate the antibody. All of these data are available within this article.

Peptide-titration immunohistochemistry
Wildtype fly brains were isolated and fixed in PFA as described elsewhere [1]. The epitope used as an antigen to generate VAChT antibody was diluted in the VAChT antibody to a 1:1 M ratio (see [1]). This peptide-antibody mixture or VAChT antibody without any peptide (control) was allowed to incubate with fly brains overnight at 4°C. Brains were stained, mounted, and imaged as described below.  Phase contrast image shows the structure and anatomy of the tissue. Each micrograph represents a single optical confocal slice. Scale bar, 20 um. Images were acquired under identical confocal microscope capture settings, post-processing was performed using identical brightness/contrast settings.

Immunostaining and confocal microscopy
Immunostaining and confocal microscopy were performed as described previously, see [1] and [2].

Funding sources
This work was supported by the National Institutes of Health (NIGMS COBRE grant 4P20GM103653-05); the National Institute on Aging (K01 grant 1K01AG049055-01A1); by a MINDS Fellowship (supported by an NIH/NINDS R25 grant); and by the National Science Foundation (EPSCoR grant).  figure) shows a strong reduction in VAChT signal compared to ChAT-Gal4,UAS-GFP/ þ control (designated as "WT" in the figure) (B) The RNAi experiment was performed using an independent UAS-RNAi construct from the Bloomington Drosophila Research Center (BDSC) (designated as "BDSC-RNAi" in the figure) and a reduction in staining is also observed compared to ChAT-Gal4,UAS-GFP/ þ (designated as "WT" in the figure). Note that baseline signal intensity is lower in B relative to A. Each micrograph represents a single optical confocal slice. Scale bar, 20 um. Images were acquired under identical confocal microscope capture settings, post processing was performed using identical brightness/contrast settings. The data are representative of three independent experiments.

Author contributions
SB contributed to the experimental design, the writing of the manuscript, and performed the experiments in Figs. 1, 2 and 3. HL directed the experimental design; prepared the figures; coordinated the writing of the manuscript; and was responsible for the intellectual direction of the paper.

Transparency document. Supplementary material
Transparency document associated with this article can be found in the online version at http://dx. doi.org/10.1016/j.dib.2017.09.008.