Tcs sp2 aobs laser scanning spectral inverted confocal microscope

For the time-lapse experiments, the embryos were extracted and dissected in cold PBS. Samples were embedded in low melting point agarose (4 %) and sectioned at 250 µm. The sections were collected using Krebs 1X medium (Krebs, glucose, NaHCO₃, Hepes 1 M 1 %, penicillin/streptomycin 1 %, Gentamicina 0.2 %) at 4 °C. The selected slice was placed in a polycarbonate membrane (MilliCell PICMORG50) with neurobasal medium and incubated during the experiment (37 °C, 5 % CO₂).
For confocal imaging, a Leica SPE-II DM5550 laser scanning confocal microscope was used. A TCS-SP2-AOBS laser scanning spectral inverted confocal microscope (fitted with temperature and CO₂ control; Leica Microsystems) was used for live imaging of brain slice culture. Images were collected every 20 min during 16 h. All the focal planes were merged to visualize the maximum projection. Videos were processed with Imaris and ImageJ software.

For the time-lapse experiments, the embryos were extracted and dissected in cold PBS. Organotypic Neural Tissue explant Cultures (ONTC) were performed as described in (Echevarría et al. 2001 ). The ONTCs were placed in a polycarbonate membrane (MilliCell PICMORG50) with neurobasal medium and incubated during the experiment at 37 ℃, and 5% CO₂.
A TCS-SP2-AOBS laser scanning spectral inverted confocal microscope (fitted with temperature and CO₂ control; Leica Microsystems) was used for live imaging of the ONTC. Images were collected every 20 min during 18 h. All the focal planes were merged to visualize the maximum projection. Videos were processed with ImageJ (FIJI) software.