2.6. Glial immunohistochemical identification
Brain coronal sections of 30 μm were obtained (Leica Microsystems CM
3050S cryostat, Wetzlar, Germany) and kept in a cryoprotectant solution
at −20°C until use. Free-floating slices were placed in a 24-well plate
and washed with 0.01M PBS. Next, the free-floating sections were blocked
with 0.1M PBS solution containing 1% BSA, 0,3% Triton X-100 for 20min
at room temperature. Afterwards, slices were washed with PBS 0.01M two
times for 5 min each and were incubated with the primary antibodies
listed in Table X overnight at 4°C. The primary antibodies were diluted
in a 0.1M PBS solution containing 1% BSA and 0.3% Triton x-100. On the
following day, the coronal slices were washed with 0.1M PBS 0.1M 2 times
for 5 min each and then incubated with the secondary antibodies listed
in Supplementary Table S1 at room temperature for 1h. Later, the
sections were washed 2 times for 5 min each with 0.1M PBS and were
incubated with 5μM Hoechst staining solution (Sigma-Aldrich, St. Louis,
MO) for 5 min in the dark at room temperature. After being washed, the
slices were mounted using Fluoromount-G (EMS, USA).