Imaging and Quantification of Protein Aggregates in Genetically Modified Drosophila Brains

Abstract

Source: Donnelly, K. M., et al. Monitoring Cell-to-cell Transmission of Prion-like Protein Aggregates in Drosophila Melanogaster. J. Vis. Exp. (2018)

This video demonstrates imaging and quantification of protein aggregates in genetically modified Drosophila brains. Brains are fixed, washed, and treated with an antifading agent before mounting. Images are captured using a confocal microscope and quantified to observe the transmission of mutant protein aggregates.

Protocol

1. Coupling Gal4- and QF-mediated Htt Transgene Expression in Drosophila

1. Collect and/or generate transgenic Drosophila melanogaster lines containing tissue-specific Gal4 or QF "drivers," as well as lines containing wild-type or mutant Htt transgenes downstream of the Gal4-UAS or QF-QUAS. Ensure that the proteins that are expressed from these transgenes are fused to fluorescent proteins or epitope-tagged to allow for differentiation of mutant and wild-type Htt transgene products in the same fly. See Figure 1.
   NOTE: We typically use exon 1 fragments of the human Htt gene. However, transgenic flies can instead be gener....

Results

Figure 1. A genetic approach for coupled expression of mutant and wild-type Htt transgenes using the QF-QUAS and Gal4-UAS binary expression systems. In "cell A," mCherry-tagged mutant Htt protein containing a pathogenic-length polyQ stretch (Q91) is expressed u.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Phosphate buffered saline (PBS), 10X, pH 7.4	ThermoFisher Scientific	AM9625	Dilute to 1X
Triton X-100	Sigma-Aldrich	T9284-1L
Kimwipes	Thomas Scientific	2904F24
20% paraformaldehyde (PFA)	Electron Microscopy Sciences	15713-S
Normal Goat Serum (NGS), filtered	Lampire Biological Laboratories	7332500	Aliquot and freeze upon receipt
Chicken anti-GFP	Aves Labs	GFP-1020	Use at 1:500 dilution
Rabbit anti-DsRed	Clontech	632496	Use at 1:2000 dilution; can recognize DsRed-based fluorescent proteins (e.g. mCherry, mStrawberry, tdTomato, etc.)
Mouse anti-Bruchpilot	Developmental Studies Hybridoma Bank	nc82	Use at 1:100 dilution; will label active pre-synaptic structures thoughout the fly brain
FITC anti-chicken	ThermoFisher Scientific	SA1-7200	Use at 1:250 dilution
Alexa Fluor 568 anti-rabbit	Life Technologies	A11011	Use at 1:250 dilution
Alexa Fluor 647 anti-mouse antibody	Life Technologies	A21235	Use at 1:250 dilution
Slowfade Gold Antifade Reagent	Life Technologies	S36936
Microscope Slides (25 x 75 x 1.0 mm)	Fisher Scientific	12-550-143
Cover Glass (22 x 22 mm)	Globe Scientific	1404-15
Dumont Biology Grade Forceps, Style 3	Ted Pella	503	use in non-dominant hand
Dumont Biology Grade Forceps, Style 5	Ted Pella	505	use in dominant hand
LAS X image analysis software	Leica
Imaris image analysis software	Bitplane