5.2.8 Chronic Damage Analysis........................
Brain slice preparation: (1) Mice underwent transcardial perfusion (40 mL PBS)
and werefixed with 4% formaldehyde 4–5 weeks after the surgery [ 28 ]. (2) Mice
were decapitated and brains were removed from the skull and set in 4%
formaldehyde for 24 h as postfixation and then PBS for 24 h to remove extra
formaldehyde. Electronics was kept inside the brain throughoutfixing process.
(3) Brains were blocked, separated into the two hemispheres and mounted on the
stage of vibratome. 50– 100 μm vibratome tissue slices (horizontal and coronal
orientations) were prepared for samples with staining for microglia, astrocytes and
nuclei. 30– 50 μm vibratome tissue slices (horizontal and coronal orientations) were
prepared for samples with staining for neurons. For samples with electronics
injected in lateral ventricle, brains were blocked and thenfixed in 1% (w/v) agarose
type I-B tofix the position of electronics in the lateral ventricle cavity and then
mounted on the stage of vibratome. 100μm vibratome tissue horizontal slices were
prepared. Coronal slices allowed for cuts in a direction along the long axis of the
injected electronics and horizontal slices allowed for cuts in a direction perpen-
dicular to the long axis of the injected device.
Chronic immunostaining for astrocytes and microglia: (1) Sections were then
cleared with 5 mg/mL sodium borohydride in HEPES-buffered Hanks saline
(HBHS) for 30 min, with 3 following washes with HBHS in 5–10 min intervals.
Sodium azide (4%) was diluted 100x in HBHS in all steps using HBHS. (2) Slices
were incubated with 0.5% (v/v) Triton X-100 in HBHS for 30 min at room tem-
perature. (3) Slices were blocked with 5% (w/v) FBS and incubated overnight at
room temperature. (4) Slices were washed four times in 30 min intervals with
HBHS to clear any remaining serum in the tissue. Slices were then incubated
overnight at room temperature with the GFAP primary antibody and rabbit
anti-Iba-1 primary antibody containing 0.2% triton and 3% serum. (5) After incu-
bation period, slices were again washed four times for 30 min with HBHS, slices
were incubated with secondary antibody Alexa Flour 546 goat anti-rat and sec-
ondary antibody Alexa Fluor 488 goat anti-rabbit, Hoechst 33342, 0.2% Triton and
3% serum overnight. (6) After thefinal washes (four for 30 min each HBHS), Slices
were mounted on glass slides with coverslips using Prolong Gold mounting media.
The slides remained covered (protected from light) at room temperature, allowing
for 12 h of clearance before imaging.
Chronic immunostaining for neuron: Slices were cleared with 5 mg/mL
sodium borohydride in HBHS for 30 min, with 3 following washes with HBHS in
5 – 10 min intervals. Then, slices were incubated with 0.5% (v/v) Triton X-100 in
HBHS for 30 min at room temperature. Next, sections were blocked with 5% (w/v)
serum and incubated overnight at room temperature. Next, slices were washed four
times in 30-minute intervals with HBHS to clear any remaining serum in the tissue.
Slices were then incubated with primary antibody NeuN in 0.3% Triton-X100 and
3% serum in PBS overnight at room temperature. After 24 h, sections were washed
four times for 30 min in PBS and then counterstained with Hoechst 33342. Prolong
5.2 Experimental 77