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As brain tissues were sliced in series sections, the counting and analysis in cell number and structures were able to be done at least from two sections for each of barrels.
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Brain tissue was sliced into ∼1-mm pieces and incubated in HBSS (Gibco) containing 20 U/ml papain (Sigma) at 34°C for 30 min. Trypsin inhibitor (Sigma) was added to a final concentration of 0.5 mg/ml and the tissue was mechanically dissociated by passing through a flame-polished Pasteur pipette.
The brain tissues were harvested and cut into 2-mm coronal slices starting 2mm from the frontal pole.
After being immersed in 20% sucrose solution and frozen in liquid nitrogen, coronal sections of the brain tissue were cut into slices of 12 μm in thickness and stored at −20°C for enzymohistochemistry morphological studies.
Fixed-cryopreserved brains were sliced at 16 μm, tissue slices were allowed to dry at least one hour at RT and processed for immunofluorescence.
Tissue samples were sliced using a cryostat at 30 µm (brains) or 12 µm (eyes).
The brain tissue was removed and successivelly sliced into 2.0 mm-thick coronal sections.
The brain tissue was then embedded in paraffin and sliced into 5 μm coronal sections containing entire SN and ST (anteroposterior levels: bregma −2.92 to −3.64 mm and +0.02 to +0.86 mm) with the reference of mouse brain atlas [ 20] for immunohistochemical study.
The entire slice of brain tissue was soaked in a form of inert glutamate, and the laser activated the chemical in precisely the area the scientists were focusing on.
The brain tissue was collected and prepared as frozen slices (20 μm) after 10%and20%0% sucrose gradient dehydration.
Using the optic chiasm as the caudal marker, a 2 mm coronal slice of brain tissue was isolated.
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