Two-Photon Microscopy for Studying Microglial Process Attraction Toward a Compound in a Mouse Brain Slice

Abstract

Source: Etienne, F. et al. Two-photon Imaging of Microglial Processes' Attraction Toward ATP or Serotonin in Acute Brain Slices. J. Vis. Exp. (2019)

This video demonstrates the use of two-photon microscopy to study the attraction of microglial processes toward an injected compound in brain slices. It provides an overview of preparing mouse brain slices, injecting a chemoattractant test compound, and imaging fluorescently labeled microglia to visualize their responses.

Protocol

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.

Results

Figure 1: Interface chamber details. (A) Outline for the 3D printing of the slice holder. The external diameter of the holder is 7 cm. (B) Interface slice holder with.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
for slice imaging
× 25 0.95 NA water-immersion objective	Leica Microsystems (Germany)	HCX Irapo
2-photon MP5 upright microscope with resonant scanners (8 kHz) and two HyD Hybrid detectors	Leica Microsystems (Germany)
Antlia-3C Digital Peristaltic pump	DD Biolab	178961	For 2-photon chamber perfusion with aCSF
Carbogen 5% carbon dioxide/95% oxygen	Air Liquide France Industrie	I1501L50R2A001
Chameleon Ultra2 Ti:sapphire laser	Coherent (Germany)
Disposable transfer pipettes , wide mouth	ThermoFischer scientific	for example : 232-11	5.8ml with fin tip, but we cut it (approx 7cm) to have a 4 mm diameter mouth
Emission filter SP680	Leica Microsystems (Germany)
Fluorescent cube containing a 525/50 emission filter and a 560 dichroic filter (for fluorescence collection)	Leica Microsystems (Germany)
Glass beaker with 50 mL of ACSF to maintain constant perfusion of the slice
Heating system	Warner Instrument Corporation	Automatic Heater Controller TC-324B	to maintain perfusion solution at 32°C
Perfusion chamber			home-made, the file for 3D printing is provided in Supplemental Material
Slice holder ("harp")			home made : hairpin made of platinum with the two branches joined by parallel nylon threads
For slice stimulation
Adenosine 5′-triphosphate disodium salt hydrate (ATP)	Sigma	A-26209	to be prepared ex-temporaneously : 1mg/ml (3mM) stock solution prepared the day of the experiment, kept at 4°C (a few hours) and diluted just before use
Fluorescein (optional)	Sigma	F-6377	use at 1 µM final
Micromanipulator	Luigs and Neumann	SM7	connected to the micropipette holde
Micropipette holder			same as for eletrophysiology
Serotonin hydrochloride	Sigma	H-9523	aliquots of 50mM stock solution in H20 kept at -20°C. 500µM solution prepared the day of the experiment.
Syringe 5mL (without needle)	Terumo medical products	SS+05S1
Transparent tubing	Fischer Scientific	11750105	Saint Gobain Performance Plastics™ Tygon™ E-3603 Non-DEHP Tubing
For image analysis
Fiji		https://fiji.sc
Icy	Institut Pasteur	http://icy.bioimageanalysis.org
Mice
CX3CR1-GFP mice	Jung et al, 2000		male or females, P3 to 2 months-old ; we have backcrossed these mice on 129sv background.
CX3CR1creER-YFP mice	Parkhurst et al 2013		male or females, P3 to 2 months-old ; we have backcrossed these mice on 129sv background.