Mounting Adult Drosophila Brains: A Method to Prepare Slides for Confocal Imaging

Overview

This video describes a method of mounting intact brains from adult flies under a bridge slide and shows the protocol performed on immunostained brains for fluorescent microscopy.

Protocol

This protocol is an excerpt from Kelly et al., Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains, J. Vis. Exp. (2017).

1. Mounting Adult D. melanogaster Brains onto Microscope Slides and Imaging

1. Build a "bridge" slide. Position two "base" coverslips roughly 1 cm apart on a positively charged slide. Ensure that the positively charged side of the slide is facing up. Adhere the coverslips to the slide with fingernail polish as shown in Figure 1A. It is usually helpful to seal the three outer edges of each base cover slip with fingernail polish to ensure mounting media does not wick under these base cover slips. Let fingernail polish dry completely (10 - 15 min) before proceeding.
2. Place the slide under the stereomicroscope and pipette the mounting media solution containing the dissected brains into the space between the two coverslips. To provide more contrast, it is useful to maneuver the gooseneck lights so that they are parallel with the bench top.
3. Remove extra mounting media from the slide using a pipette, being careful not to pipette the brains off the slide.
4. Wick away extra mounting media. This will allow the brains to be positioned more precisely during the next step.
5. Using a pair of forceps and the stereomicroscope, position the brains on the slide in a grid pattern with antennal lobes facing up.
6. Place a cover slip (the "bridge") over the brains (Figure 1A). Use fingernail polish to seal the sides of the bridge cover slip where they contact the "base" cover slips.
7. Using a p200 pipette, slowly fill the center cavity under the bridge with fresh mounting media (Figure 1B). Place one drop at a time on the open edge of the center coverslip and allow the mounting media to wick under the center bridge coverslip. Continue until the entire cavity is filled with mounting media, then seal the top and bottom with clear fingernail polish.
8. Once the fingernail polish is dry, image the slides immediately or store in a lightproof tight slide box at -20 °C.
9. Within one week, image brains using a laser-scanning confocal microscope with excitation lasers and filter cubes appropriate to the chosen fluorescent secondary antibodies. Z-stack images of mushroom body neurons are typically obtained using 20X or 40X objectives. Imaging of retinal photoreceptor neurons may require higher magnification.

Representative Results

Figure 1: Mounting brains in preparation for immunofluorescence imaging. (A) Brains are mounted on SuperFrost Plus slides with a bridge cover to prevent flattening of the brains. Two "base" cover slips are adhered to a Superfrost Plus positively-charged slide with clear fingernail polish and dissected brains are then placed on the slide between them. A clear "bridge" cover slip is placed above the brains and adhered to the base cover slips. (B) Once the fingernail polish has dried, Vectashield is slowly pipetted under the bridge to preserve the fluorescence of the secondary antibody. Clear fingernail polish is then used to seal the top and bottom of the "bridge" cover slip. Please click here to view a larger version of this figure.

Materials

Name	Company	Catalog Number	Comments
Microdissection forceps/tweezers	Ted Pella	505-NM
VectaShield	Vector Labs	H-1000
SuperFrost Plus Slides	ThermoFisher	99-910-01
Coverslips	ThermoFisher	12-553-454
fingernail polish	Electron Microscope Services (EMS)	72180
stereomicroscope	Leica S6D with KL300 LED light source
Kimwipe	Fisher	06-666