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The complex 3-D organization of the mutated brain sections was made plainly visible.
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3 mm coronal brain sections were made and embedded in paraffin wax.
Brains were stored at 4°C. 75 µm-thick sagittal brain sections were made from transgenic 5HT3A-EGFP mice (P15) using a vibratome (Leica VT1000S).
Frozen brains were embedded in Tissue Freezing Medium (Leica Instruments, Germany) at −20°C, and 6 8 µm-thick sagittal brain sections were made using the vibratome Microm HM500 (MICROM International, Germany).
Then, 20 μm cryostat (Thermo Fisher Scientific) coronal brain sections were made.
More specifically, consecutive 2-, 1- and 2-mm-thick brain sections were made, starting at 2 mm from the olfactory bulb.
From the brain 5 coronal sections were made, starting from the frontal lobe to the cerebellum as previously recommended [ 9].
For volumetric analysis of contusion size, mice were perfusion fixed as described above and entire brains cryosectioned; 10 µm sections were made every 0.5 mm.
Brains were postfixed, and 100-µm vibratome sections were made.
Brains were frozen, 25 µm thick frontal sections were made on a Leica cryostat and processed as described previously (Goldschmidt et al. 2004, 2010).
In the case of immunoelectron microscopy, 30-μm-thick sections were made from mouse brains fixed with a mixture of 2% paraformaldehyde and 0.1% glutaraldehyde in 0.1 M phosphate buffer.
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