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).
   1. Submerge a dissected brain in fibrinogen working solution (fibrinogen final concentration ~3.3 mg/mL, 10.5 µL per brain) as follows:
   2. Aspirate 3.5 µL of schneider active medium (SAM)/antibiotics. Aspirate a dissected brain from the dissection well into the same pipette tip without dispensing the SAM/antibiotics medium in the tip.
   3. Dispense the brain and the medium on the lid of a sterile Petri dish. Add another 3.5 µL of SAM/antibiotics and 3.5 µL of warm fibrinogen/SAM 10 mg/mL solution to the drop containing the brain. Gently mix the solution around the brain by pipetting with a 20 µL pipette tip.
      NOTE: Using a pipette rather than forceps in this step avoids stressing the brain.
   4. Take 2 µL of the fibrinogen working solution from the drop and apply it as a small circle on the coverslip of the glass-bottom dish. Add 0.8 µL of thrombin and mix very quickly with a pipette tip. The fibrinogen is converted to fibrin and starts to polymerize.
   5. Take the brain that sits in the fibrinogen working solution (step 1.1) between a pair of forceps and position it on the matrix once a white matrix of fibrin is visible. Orient the brain anterior side down (for imaging clock neurons). Push it down as close as possible to the cover glass. The fibrin clot is composed of layers of fibrin. Pick one layer and fold it on top of the brain with small and gentle movements without detaching the matrix.
      1. Repeat 2 to 3 times from different parts of the clot to stabilize the brain.
         NOTE: When handling the brain with forceps, be careful not to dry it or impose physical stress on it.
   6. Add 20 µL of SAM/antibiotics on top of the embedded brain to stop the action of thrombin.
   7. Repeat the procedure to mount the remaining brains. Up to five to six brains can be embedded in a glass-bottom dish.
   8. For long-term live imaging, add 600 µL of SAM/antibiotics in the inner well (the glass part). Position a pre-cut Polytetrafluoroethylene (PTFE) membrane on top of the medium and stick it to the vacuum grease applied on the plastic part of the bottom (Figure 1A).

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