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were again desiccated for 20 min prior to analysis using a
Solarix 7T FTICR mass spectrometer (Bruker Daltonics)
and FlexControl. Spectra were acquired across both tissue
sections (both tissues were acquired in one imaging run)
operating in positive ion mode to detect lipid species of
interest in the m/z 200-1200 range with a SmartBeam II
laser operating at 1000 Hz, a laser spot size of 25 μm, and a
lateral resolution of 100 μm, collecting 400 shots per pixel,
with a resolution of ~70,000 at m/z 800. Example spectra
from two regions of interest (ROIs), along with a list of
achievable resolutions, are included in the supplementary
data (Suppl. Figs. S1 and S2). Data were reduced to a 1
ICR reduction noise threshold, and normalized using rootmean-square vector normalization.25 Data were visualized
using FlexImaging (Bruker Daltonics). All the GSLs identified in this study contain a dihydroxy group on the sphingosine backbone. The lipids were identified by accurate mass,
cross-referenced to the Lipid Maps database, and confirmed
with collision-induced dissociation (CID) where indicated
(see Suppl. Figs. S5-S7).

Glycosphingolipids Analyses by LC-ESI-MS/MS
The left brain hemisphere was sliced sagittally and dissected by region, collecting the olfactory bulb (OB), cortex
(CO), midbrain (MID), brain stem (BS), and cerebellum
(CB). GSLs were extracted for LC-ESI-MS/MS quantitative analysis as described previously.24 Quantitation of
GSLs, including GluCer/GalCer, LacCer, sulfatide, and
gangliosides, was carried out using a Waters (Milford, MA)
Quattro Premier triple quadrupole mass spectrometer interfaced with the Acquity UPLC system. The triple quadrupole
mass spectrometer was operated under unit resolution.
Optimized parameters for GSLs were determined with individual standard compounds. Like MALDI IMS results, all
the GSLs identified in this study contain a dihydroxy group
on the sphingosine backbone. The isomeric analysis of
GluCer and GalCer was achieved by resolving the two isomers on a silica column (Phenomenex 2.1*250 mm) running under the hydrophilic interaction liquid chromatography
(HILC) mode. Five standards (C16, C18, C24:1 GluCer and
C16, C24:1 GalCer) were used, and C12 GluCer and C12
GalCer were used separately as internal standards.
Lactosylceramides (LacCers) and gangliosides were analyzed with an Acquity UPLC CSH C18 column (2.1 × 100
mm, 1.7 μm) (Waters). Standards of gangliosides GM1,
GM2, and GM3 and C16 LacCer and C24 LacCer from commercial sources were used to construct calibration curves,
and C12 LacCer was used as an internal standard. For quantification, the precursor and product ion pairs for multiplereaction monitoring (MRM) were either selected by MS/MS
spectra of standards or calculated theoretically if the standard
for a given ceramide length was not readily available.
Analysis of gangliosides was performed under negative

SLAS Discovery 22(10)
mode, while LacCers were under positive mode. Composition
of individual species in the standards was determined by the
ratio of standard infusion ion intensity or by description from
the supplier. Sulfatides were analyzed under negative ion
mode by detection of their parent deprotonated ion and common product ion of m/z 97. C16 and C24 sulfatide standards
were used to construct calibration curves, and C12 sulfatide
was used as an internal standard. Quantification of GSL species with various fatty acid chain lengths was realized by
using the curve of each GSL species with the closest number
of chain length. GSL levels in the tissues were normalized to
wet tissue weight.

Immunohistochemistry
The left brain hemisphere was sectioned sagittally and fixed
in 4% paraformaldehyde, processed for frozen blocks, and
sectioned. Immunohistochemistry studies with anti-CD68,
anti-glial fibrillary acidic protein (GFAP), and anti-p62 staining were performed using the DISCOVERY XT Staining
Module on a BenchMark XT Automated IHC/ISH slide
staining system (Ventana Medical System) at the Pathology
Research Core at the Cincinnati Children's Hospital Medical
Center.26 Frozen tissue sections were incubated with each
antibody diluted in DISCOVERY Antibody diluent, followed
by biotinylated secondary antibody. Detection was performed
using the Ventana IHC DAB Map Kit. Tissue sections were
counterstained with hematoxylin and were analyzed by light
microscopy. Images were acquired by digital scanning using
AperioImageScope v2.

Results
Mapping and Quantitation of Glycosphingolipids
by Brain Region
For GSL imaging analysis, the brains from GD (4L/PS-NA)
and control mice were collected and analyzed following the
workflow summarized in Figure 1A.21 Briefly, brains from
4L/PS-NA and control mice were split sagittally into two
(left and right) hemispheres. Right hemispheres were analyzed using MALDI FTICR IMS analysis by sectioning the
brains and placing both the control and GD brains on the
same slide (Fig. 2A). In an effort to collect more comparable data, imaging runs included both tissues. GSL lipid species were identified by mass and compared across conditions
using relative ion intensities. An abundant brain lipid phosphatidylcholine (PC) 32:0 (Fig. 2B) was included to demonstrate consistent signal across the two different brains.
However, differential distributions can be seen in the grey
versus white matter, which is consistent with data shown
previously.23 Using this approach, many different hexosylceramides (HexCers) were detected (Fig. 2C). Of these,
HexCer d18:1/18:0 + Na, HexCer d18:1/20:0 + Na, and

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