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Method Article

Immunofluorescence Staining of Drosophila Brains for the Single-Cell Imaging of Glial Cells

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July 8th, 2025

In This Article

Abstract

Source: Batelli, S., et al. Application of MultiColor FlpOut Technique to Study High Resolution Single Cell Morphologies and Cell Interactions of Glia in Drosophila. J. Vis. Exp. (2017).

This video demonstrates the immunofluorescence staining of a transgenic Drosophila brain for single-cell imaging of glial cells. The brain expresses membrane-targeted proteins fused with different combinations of antigenic epitopes under the control of a glial-specific recombinase system. The isolated and fixed brains are stained with primary antibodies specific to the antigenic epitopes, followed by fluorophore-conjugated secondary antibodies. This labeling enables the simultaneous imaging of different glial cell subpopulations across various brain regions.

Protocol

1. Preparing Flies for MultiColor FlpOut (MCFO) Experiments

NOTE: The MCFO technique refers to a modified version of the so called Flp-mediated stop cassette excision (FlpOut). Transgenic MCFO flies carry a heat shock promoter (hsp)-Flp recombinase and different reporters under upstream activation sequence (UAS) control. Each reporter consists of a common backbone of a myristoylated (myr) super folder green fluorescent protein (sfGFP), in which 10 copies of an epitope tag (e.g., HA, FLAG or V5) have been inserted. The resulting non-fluorescent proteins are named "spaghetti monster GFPs" (smGFPs) and ....

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Results

Genetic cross diagram; UAS/GAL4 system; illustrates heat shock-induced gene expression regulation.
Figure 1: Crossing scheme for the MCFO technique. Schematic of the MCFO technique. Homozygousvirgins carrying the MCFO cassette and the hsp-Flp (hsp-Flp; 10XUAS-FRT-stop-FRT-myr-smGFP-HA,10XUAS-FRT-stop-FRT-myr-smGFP-FLAG, 10XUAS-FRT-stop-FRT-myr-smGFP-V5.......

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Disclosures

No conflicts of interest declared.

Materials

List of materials used in this article
NameCompanyCatalog NumberComments
Water bathGrantGD100
PCR tubesSarstedt72.737.002
ForcepsDumont11251-20
Dissecting dish 30 mm x 12 mmmElectron Microscopy Sciences70543-30Glass dissection dish
Pyrex 3 Depression Glass Spot PlateCorning7223-34Glass dissection plates
Sylgard BlackSYLGARD, Sigma-Aldrich805998home made with charcoal
ExpressFive S2 cell culture mediumInvitrogen10486-025
20% PFAElectron Microscopy Sciences15713
Triton X-100Roth3051.3
Normal goat serumJackson Laboratories005-000-121
Normal donkey serumJackson Laboratories017-000-121
Bovine Serum AlbuminSigmaA9647
Rabbit HA-tagCell SignalingC29F4Primary antibody, dilution 1:500
Rat FLAG-tagNovus BiologicalsNBP1-06712Primary antibody, dilution 1:100
Mouse V5-tag:DyLight 549AdSerotec411Conjugated antibody, dilution 1:200
anti-rabbit AlexaFluor 488InvitrogenA11034Secondary antibody, dilution 1:250
anti-rat DyLight 647Jackson Laboratories712-605-153Secondary antibody, dilution 1:100
Vecta ShieldVector LaboratoriesH-1000
SlowFate GoldInvitrogenS36937
Secure Seal SpacerGrace BiolabsContact company for ordering
Microscope cover glass 22 X 60 mmMarienfeld101152
Microscope cover glass 22 x 22 mmRothH874
Stereo Microscope, Leica MZ6Leica
Confocal laser scanning microscope LSM710Zeiss
ImmersolZeiss518 FImmersion oil for fluorescence-microscopy, halogen free
ImmersolZeissW 2010Immersion fluid for water-immersion objectives, halogen free
R56F03-GAL4 (EG)Bloomington Stock Center39157GAL4 driver
R86E01-GAL4 (ALG)Bloomington Stock Center45914GAL4 driver
hspFlpPestOpt; UAS-FRT-stop-FRT-myr-smGFP-HA, UAS-FRT-stop-FRT-myr-smGFP-FLAG, UAS-FRT-stop-FRT-myr-smGFP-V5Bloomington Stock Center64085UAS reporter (https://bdscweb.webtest.iu.edu/stock/misc/mcfo.php)
Multi Time MacroZeissSoftware for automated scanning

Tags

Drosophila BrainPrimary AntibodySecondary AntibodyFluorophore ConjugationTissue FixationMounting MediumCell Specific Recombinase