Dissection and Mounting of Fruit Fly Larval Brain Explants

Samenvatting

Source: Sheng, C., et al. Time-lapse Live Imaging and Quantification of Fast Dendritic Branch Dynamics in Developing Drosophila Neurons. J. Vis. Exp. (2019).

This video demonstrates the dissection and mounting of brain explants from Drosophila larvae. The central nervous system (CNS), attached to eye disks, is extracted from the larvae and then mounted on a glass slide in a physiological saline solution.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Dissecting and Mounting Larval Brain Explants

1. Dissect larval brains in the physiological external saline solution (120 mM NaCl, 4 mM MgCl₂, 3 mM KCl, 10 mM NaHCO₃, 10 mM Glucose, 10 mM Sucrose, 5 mM N-[Tris(hydroxymethyl)methyl]-2-aminoethanesulfonic acid (TES), 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 2 mM Ca²⁺, pH 7.2) under a dissection microscope (4.5x magnification power) with two pairs of #5 standard tip dissection forceps (11 cm). Use one pair of forceps to hold the larval body in place and the other to carefully dissect out the brain. Preserve the eye disks, brain lobes and the ventral nerve cord. Remove attached muscles to minimize sample movements during imaging.
2. Prepare a glass slide (25 x 75 x 1.0 mm³) and use a syringe to draw a square chamber with vacuum grease.
3. Add 20 µL of external saline solution to the square chamber with grease barriers.
4. Transfer dissected larval brains into the chamber on the glass slide using forceps. Adjust the position of the brains under the dissection scope to ensure the dorsal side faces up.
5. Cover the chamber with a glass cover slip (22 x 22 x 0.15 mm³). The larval brain is now mounted on the slide within a chamber filled with the external saline solution (Figure 1B).
   NOTE: Pressing gently on the coverslip confines the brains and reduces sample drifting in the subsequent imaging session.

Resultaten

Figure 1: Workflow of the dendrite dynamics imaging and quantification protocol. (A) The protocol contains six steps covering sample preparation, image collection, image processing and semi-automated quantification of dendrite dynamics....

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
high vacuum grease	Dow Corning	79751-30
Microscope Cover Glass	Fisher Scientific	12-544-E
Superfrost Plus Microscope Slides	Fisher Scientific	12-550-15
Reagents
Glucose
HEPES
KCl
MgCl2
NaCl
NaHCO3
PBS
Sucrose
TES