eBook: Advancing Neuroscience Research - 25

Biosystems, C13001) and then placed in the ETC
Chamber of the X-CLARITY™ Tissue Clearing System
(Logos Biosystems, C10101). Electrophoretic
Tissue Clearing Solution was circulated through the
chamber and 0.7 A was applied across the brains
for 6-12 hours at 35°C. After clearing, brains were
washed with PBS overnight at room temperature
to remove residual SDS.
Technical Tips
- Endogenous FP signals can degrade at higher
temperatures. To prevent excessive Joule heating,
optimize the electric current applied during electrophoresis.
Use lower currents to minimize high
temperature increases. This may also affect clearing
time.
Immunostaining
Clarified brains were cut into 1 mm slices or hemispheres
for immunostaining. Brain samples were
incubated in anti-Collagen IV (1:100, Abcam) in 6%
BSA, 0.2% Triton X-100, 0.01% sodium azide in 0.1
M PBS for 24 hours at 37°C with gentle shaking.
After 24 hours, samples were washed with PBST
(0.2% Triton X-100, 0.01% sodium azide in 1X PBS)
overnight at 37°C. Samples were then incubated
with donkey anti-rabbit Cy3 Fab fragment (1:250,
Jackson ImmunoResearch) and TO-PRO-3 Iodide
(1:1000, ThermoFisher Scientific) in 6% BSA, 0.2%
Triton X-100, 0.01% sodium azide in 0.1 M PBS for
24 hours at 37°C with gentle shaking. Samples were
washed with PBST overnight at 37°C.
Refractive index matching
and imaging
Stained samples were rinsed with distilled water for
5 minutes and immersed in X-CLARITY™ Mounting
Solution (Logos Biosystems, C13101) for 1 hour at
room temperature with gentle shaking. The solution
was replaced and samples were incubated
for another 1-2 hours. For confocal imaging, brain
slices were placed in a 35 mm glass-bottomed dish
(SPL Life Sciences) with fresh mounting solution
and imaged with a LSM 710 (Carl Zeiss) using the EC
Plan-Neofluar 10x/0.3 objective. ZEN software (Carl
Zeiss) was used to process the images. For light
sheet imaging, brain hemispheres were placed in
an imaging chamber with fresh mounting solution
and imaged with a Lightsheet Z.1 (Carl Zeiss) using
a EC Plan-Neofluar 5x/0.16 objective lens. ZEN (Carl
Zeiss) software was used to process confocal images.
Amira 3D software (FEI) was used to process
light sheet images and render a 3D video.
Technical Tips
- Fluorescence imaging should be done immediately
after RI matching to minimize the loss of fluorescence
signal. Stained samples can be stored in
the mounting solution for up to 3 days without significant
signal loss, but for long term storage, place
samples in PBS at 4°C. - For more information, read
" The X-CLARITY™ Mounting Solution: an improved
RI matching solution for tissues cleared
by the X-CLARITY™ Tissue Clearing System " .
Thy1-YFP mouse brain slices cleared with the X-CLARITY™
systems and reagents.Thy1-YFP (green), Anti-Collagen IV (red),
TO-PRO-3 (blue)
25

http://logosbio.com/download/pdf/TheX-CLARITYMountingSolution-animprovedRImatchingsolutionfortissuesclearedbytheX-CLARITYTissueClearing2.pdf http://logosbio.com/download/pdf/TheX-CLARITYMountingSolution-animprovedRImatchingsolutionfortissuesclearedbytheX-CLARITYTissueClearing2.pdf http://logosbio.com/download/pdf/TheX-CLARITYMountingSolution-animprovedRImatchingsolutionfortissuesclearedbytheX-CLARITYTissueClearing2.pdf

eBook: Advancing Neuroscience Research

Table of Contents for the Digital Edition of eBook: Advancing Neuroscience Research