Immunostaining of Larval and Adult Nervous Systems

Larval and adult immunostaining were performed as previously described (Certel and Thor 2004 (link); Petersen and Stowers 2011 (link)). Primary antibodies and dilution factors: The SYN (3C11) mAb 1:25 developed by E. Buchner (Klagges et al. 1996 (link)) was obtained from the Developmental Studies Hybridoma Bank, created by the NICHD of the NIH and maintained at The University of Iowa, Department of Biology, Iowa City, IA 52242. Mouse anti-vGlut (Banerjee 2021 (link)) 1:10; Rabbit anti-Syt (Littleton et al. 1993 (link)) 1:1,000; Mouse anti-mCherry (Biorbyt orb256058) 1:200; Rabbit anti-mCherry (Abcam ab213511) 1:500, Rabbit Abfinity anti-GFP (Thermo-Fisher) 1:400, Rabbit anti-HA (Cell Signaling C29F4) 1:500, and Rat anti-FLAG (Novus NBP1-06712) 1:200. Secondary antibodies and dilution factors: Donkey anti-Rat Alexa 488 (Jackson Immunoresearch 712-546-153) 1:400, Donkey anti-Mouse Alexa 488 (Jackson Immunoresearch 715-545-151) 1:400, Goat anti-Rabbit Alexa 488 (Thermo-Fisher A32731) 1:200, Donkey anti-Mouse JF549 (Novus NBP1-75119JF549) 1:200, Goat anti-Rabbit JF549 (Novus NBP1-72732JF549) 1:200, Goat anti-Rabbit JF646 (NBP1-72732JF646). Larval muscles were stained with Phalloidin 405 (Biotium CF405M).

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Certel S.J., Ruchti E., McCabe B.D, & Stowers R.S. (2022). A conditional glutamatergic synaptic vesicle marker for Drosophila. G3: Genes|Genomes|Genetics, 12(3), jkab453.

Publication 2022

Rabbit Abfinity anti-GFP Rabbit anti-HA Rat anti-FLAG Donkey anti-Rat Alexa 488 Donkey anti-Mouse Alexa 488

Adult Antibodies Dilution Donkey Goat Hybridoma Larval Mouse Muscles Novus Phalloidin Rabbit

Corresponding Organization : Montana State University

Other organizations : University of Montana, École Polytechnique Fédérale de Lausanne

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Variable analysis

independent variables

Primary antibodies and dilution factors

dependent variables

Immunostaining patterns of synaptic proteins (SYN, vGlut, Syt, mCherry, GFP, HA, FLAG) in larval and adult tissues

control variables

Immunostaining procedures as previously described in Certel and Thor 2004 and Petersen and Stowers 2011
Dilution factors for primary and secondary antibodies

controls

Positive control: Staining with known synaptic markers (SYN, vGlut, Syt)

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