Computational protocol: Postembryonic lineages of the Drosophila ventral nervous system: Neuroglian expression reveals the adult hemilineage associated fiber tracts in the adult thoracic neuromeres

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Protocol publication

[…] Tissues were dissected in PBS (phosphate‐buffered saline, pH 7.8) and fixed in 4% buffered formaldehyde for 1 hour at room temperature. Fixed tissues were washed in PBS‐TX (PBS with 1% Triton X‐100, Sigma, St. Louis, MO), incubated in 10% normal donkey serum (Jackson ImmunoResearch, West Grove, PA; Cat. no. 017‐000‐001 RRID:AB_2337254) for up to 6 hours, and then in 1:1,000 rabbit anti‐GFP (Molecular Probes (Invitrogen), Eugene, OR; Cat. no. A11122 RRID:AB_221569), 1:40 rat anti‐N‐cadherin (Developmental Studies Hybridoma Bank, Iowa City, IA; Cat. no. DN‐Ex 8 RRID:AB_528121), and 1:40 mouse anti‐neuroglian (Developmental Studies Hybridoma Bank; Cat. no. BP 104 anti‐neuroglian RRID:AB_528402) overnight at 4°C. Tissues were washed in PBS‐TX and incubated with 1:500 AlexaFluor 488‐conjugated donkey antirabbit (Jackson ImmunoResearch; Cat. no. 715‐545‐151 RRID:AB_2341099), AlexaFluor 594‐conjugated donkey antimouse (Jackson ImmunoResearch; Cat. no. 715‐585‐151 RRID:AB_2340855), and AlexaFluor 647‐conjugated donkey antirat (Jackson ImmunoResearch; Cat. no. 712‐605‐153 RRID:AB_2340694) overnight at 4°C. This is a standard antibody protocol we use for all our studies. The N‐cadherin staining reveals the fine structure of the neuropil but the information provided by this channel was not used in this study. Tissues were washed in PBS‐TX, mounted onto poly‐lysine‐coated coverslips, dehydrated through an ethanol series, cleared in xylene, and mounted in DPX mountant (Sigma‐Aldrich). Nervous systems were imaged on either a Zeiss LSM 510 or Zeiss 710 confocal microscope at either 40× or 63× with optical sections taken at either 1 μm or 2 μm intervals. LSM files were contrast‐enhanced as necessary. z‐projected images were created using ImageJ (, Z sections were made with Vaa3D (Peng et al., ) ( and montages made in Adobe Photoshop (San Jose, CA). […]

Pipeline specifications

Software tools ImageJ, Vaa3D
Applications Laser scanning microscopy, Microscopic phenotype analysis
Organisms Drosophila melanogaster
Diseases Nervous System Malformations