Embedding Drosophila Brain in a Matrix for Time-Lapse Imaging of the Circadian Clock Neurons

Abstract

Source: Sabado, V., et al., Nagoshi, E. Single-cell Resolution Fluorescence Live Imaging of Drosophila Circadian Clocks in Larval Brain Culture. J. Vis. Exp. (2018).

This video demonstrates the procedure to embed the Drosophila brain in a fibrin matrix, followed by time-lapse fluorescence imaging of circadian clock neurons to analyze rhythmic activity synchronized with light/dark cycles.

Protocol

1. Embedding of brain explants in a 3D matrix and mounting (~20 min).

Results

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
KH2PO4	Sigma-Aldrich	P5655	I am not sure they are exactly the same ones we have in the lab. I chose "suitable for insect cell culture" whenever available
CaCl2	Sigma-Aldrich	C7902
MgSO4.7H2O	Sigma-Aldrich	230391
NaCl	Sigma-Aldrich	S5886
NaHCO3	Sigma-Aldrich	S5761
D-(+) Glucose	Sigma-Aldrich	G7021
Yeast extract	Sigma-Aldrich	Y1000
Insulin	Sigma-Aldrich	I0516-5ML
Penicillin-Streptomycin	Sigma-Aldrich	P4333
BIS-TRIS	Sigma-Aldrich	B4429
L-(−)-Malic acid	Sigma-Aldrich	M7397
D-(+)-Trehalose dihydrate	Sigma-Aldrich	T0167
Succinic acid	Sigma-Aldrich	S9512
Fumaric acid	Sigma-Aldrich	F8509
α-Ketoglutaric acid	Sigma-Aldrich	K1128
Non-heat-inactivated, Foetal Calf Serum (FCS) Mycoplasma and Virus screened	BioConcept Ltd. Amimed	2-01F30-I
HEPES-KOH, pH 7.4	E&K Scientific Products	EK-654011
KCl	Sigma-Aldrich	P5405
NaH2PO4	Sigma-Aldrich	S5011
Sucrose	Sigma-Aldrich	S7903
Fibrinogen from bovine plasma	Calbiochem (Merck)	341573-1GM	CAUTION: Harmful by inhalation, in contact with skin and if swallowed. Manipulate under laminar flow
Thrombin from bovine plasma	Sigma-Aldrich	T9549	CAUTION: Health Hazard, use gloves
PDF, NH2-NSELINSLLSLPKNMNDA-OH	Chi Scientific	custom made
Vaccum grease	Sigma-Aldrich	18405
35 mm Dish, No. 1.5 Coverslip, 20 mm Glass Diameter, Uncoated	MatTek	P35G-1.5-20-C
Corning Falcon Easy-Grip Tissue Culture Dishes	Fisherscientific	08-772A
Sterile 500 mL Steritop-GP 33 mm threaded bottle top filter, 0.22 μm	Millipore	SCGPS05RE
Polytetrafluoroethylene (PTFE) film	Dupont	200A Teflon FEP Film
Millex-HV Syringe Filter Unit, 0.45 µm, PVDF, 33 mm, gamma sterilized	Millipore	SLHV033RS
Millex-GV Syringe Filter Unit, 0.22 µm, PVDF, 33 mm, gamma sterilized	Millipore	SLGV033RS
Three-well glass dissection dish	Any company
Fine forceps, size 5, Dumont	Fine Science Tools	11254-20
Tandem scanner inverted TCS SP5 confocal microscope, with resonant scanner and HyD photo-multiplier detectors	Leica microsystem CMS GmbH
Temperature control chamber	Life Imaging Services	The CUBE & BOX temperature control system, custom designed
Stage-top humidity controller	Life Imaging Services	custom made
Water Immersion Micro Dispenser: dispenser, extended micro-pump MP6 series and Autoimmersion Objective Controller software	Leica microsystem CMS GmbH
SUM-stack creation and 3D correction drift plugin	ImageJ software
10x iterative deconvolution	AutoQuant and Imaris software