SBRI Stem-cell and Brain Research Institute - France (Lyon)

Technical Facilities : Histology and microscopy

Composition of team / September 1, 2017

Coordinator : Pierre Misery

  • Pierre Misery - Engineer (IE), 25%
  • Camille Lamy - Engineer (IE), 20%
  • Guillaume Marcy - Engineer (IE), 30%
  • Veronique Cortay - Technician (TR), 20%

Description Equipment

This core facility includes a histology lab (cryostats, freezing microtomes, etc), 4 ExploraNova automated imagery stations for neuronal cartography, two Leica confocal microscopes.

Histology labs

The laboratory houses a fully equipped histology lab with chemicals and glassware. The histology lab has linear hooded mounting desks for mounting sections and freezers and refrigerators for storing antibodies, and appropriate reagents for immuno-histochemistry. The histology lab is equipped with large microtome’s for cutting NHP brains.
The Microm HM 440 E Microtome is an ergonomically-designed sliding microtome. It employs electronic control systems and high-precision mechanics to achieve optimal section quality and reproducibility of paraffin and frozen specimens. It can accommodate specimen sizes up to 80 x 60 mm, and can cut sections ranging from 1 to 100 microns.

We use a Vibratome (Leica VT1000S) which is an instrument using a vibrating razor blade to cut through tissue. The vibration amplitude, the speed, and the angle of the blade can all be controlled. We only use it to section postnatal and adult mouse fixed brains embedded in low gelling temperature agarose.  Individual sections of 50 µm are collected with a fine brush and transferred to a multiwall plates for staining.

Microscope facilities

ExploraNova automated imagery stations for neuronal cartography
To explore projection pathways, we used 4 fluorescent microscopy stations running on exploranova software (Mercator). Analysis consist on the recording of the XY coordinates of very large numbers of neurons using a digitalized image system.
2D cartography of neuronal labelling are used to reconstruct 3D (Map 3D) map to obtain accurate data on individual neuron pathways length, important for modelling networks of the cerebral cortex.
One of the station, using the software morphostrider (Explora Nova), allows automatic 2D reconstructions of large and high-defined images with multiple wawelenghts.

1 / Microscope Leica Dialux 22 with fluorescence

Objectives : x2,5 x10 x25 x50 x100
Filter : D
Camera Sony DXC950-P
Mercator expert software

2 / Microscope Leica DMRE with fluorescence

Objectives : x1,6 x5 x10 x20 x25 x40
Camera Jenoptik ProgRes CF
Filters : XF102-2, N2-1, XF100-2, D, XF13-2, XF103
Mercator expert software.

3 / Microscope Leica DM5500 B with fluorescence.

Objectives : x2,5 x10 x20 x40.
Filters : A, D, Y5, I3 and N2-1.
Camera Jenoptik ProgRes CF.
Mercator expert, Map3D and  Morphostrider Software.

4 / Microscope Leica DM6000 with fluorescence

Objectives : x2,5 x10 x20 (dry) x20 (imm) x63 x100
Filters : TX2, D, DY, FB, I3, XF32, XF08
Camera Jenoptik ProgRes CF.
Mercator expert software.

Confocal microscopy

1 / LEICA SP5

The microscope is an upright DM6000CS SP5 from LEICA equipped with four laser sources.

  • Diode laser 405 nm
  • Argon laser, 458, 476, 488, 514 nm
  • He Ne 543 nm
  • He Ne 633 nm

Microscope with motorized objective nosepiece.

  • 10x/0.30 HC PL Fluotar, Dry
  • 20x/0.70 HC PL APO, oil immersion
  • 40x/1.25 HCX-PL APO, oil immersion
  • 60x/1.32 PL APO, oil immersion motorized stage.

Software: Leica LAS AF

 

2) LEICA TCS SPE:

The microscope is an upright DM5500Q SPE from LEICA equipped with three solid state laser sources.

  • 488 nm
  • 561 nm
  • 633 nm

Microscope with non-motorized objective nosepiece.

  • 10x/0.30 HCX PL Fluotar, Dry
  • 20x/0.70 HC PL APO, Dry
  • 40x/1.25 HCX-PL APO, oil immersion
  • 60x/1.40 PL APO, oil immersion
  • 100x/1.40 HCX-PL APO, oil immersion

Motorized stage.
Software: Leica LAS X