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Page 4 of 9 Klugmann et al. Rare Dis Orphan Drugs J 2023;2:8 https://dx.doi.org/10.20517/rdodj.2023.05

PVDF membrane is incubated in blocking solution (4% milk powder and 0.1% Tween in PBS) for at least
one hour at room temperature. Primary antibodies are then applied in blocking solution overnight at 4 °C.
Following three 10 min washing steps with PBS-T (0.1% Tween in PBS), the membrane is probed with
secondary HRP-coupled antibodies in blocking solution for 1 h at room temperature and washed three
times again in PBS-T, each 10 min. Afterwards, the membrane is developed using Clarity Western ECL
substrate (Bio-Rad #170-5060) and subsequently imaged employing the Bio-Rad ChemiDoc MP imaging
system. Protein bands are quantified using the densitometry function in ImageJ and AspRS protein levels in
different brain regions are normalized to the housekeeping proteins β-Actin and GAPDH. A comprehensive
AspRS expression study comparing protein levels across different regions of the human brain was
[12]
conducted following the protocol described here . This study found significantly higher AspRS levels in
the cerebellum compared to the other brain regions analyzed.

Quantitative real-time PCR
For qRT-PCR analysis, the tissue powder is resuspended in 350 µL (for up to 20 mg of tissue) or 600 µL (for
20-30 mg of tissue) buffer RLT (from the Qiagen RNeasy MiniKit #74106) plus 1% β-mercaptoethanol and
homogenized using a pellet pestle attached to the Kontes pellet pestle motor. Afterwards, the lysate is
centrifuged for 3 min at full speed in a conventional benchtop centrifuge and the supernatant is transferred
to a fresh tube. RNA extractions are performed using the RNeasy MiniKit (Qiagen #74106) following the
manufacturer’s instructions including an on-column DNase digest using the RNase-free DNase kit (Qiagen
#79254) to get rid of any genomic DNA. At the end of the extraction procedure, the RNA is eluted from the
column in 50 µL RNase free water and the concentration is determined using the NanoDrop microvolume
spectrophotometer. An equal amount of RNA from each brain region is used for cDNA synthesis using the
High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems #4368813) in a conventional
thermocycler. Afterwards, qRT-PCR can be performed in any suitable real-time PCR machine employing
specific TaqMan probes. Data is analyzed using the comparative ΔΔCT method for relative quantification of
DARS1 expression normalized to the housekeeping gene β-Actin. Using this method, DARS1 mRNA levels
were analyzed across different human brain regions, revealing a significant enrichment in the cerebellum in
line with the AspRS protein data .
[12]

Immunohistochemistry
Formalin-fixed and paraffin-embedded sections were received on glass slides from the New South Wales
Brain Bank. The sections were cut at 4 μm thickness to ensure adequate penetration of antibodies. Brain
sections included the motor cortex, hippocampus, and cerebellum, which were all cut in the coronal plane,
and sections from the brainstem (pons), which were cut in the transverse plane.

Sections for immunofluorescent labelling [Figure 1] are incubated in a 60 °C oven for 2 h to melt the
paraffin surrounding the tissue before placing the slides in xylene for 2 × 10 min to ensure complete
solubilization and removal of residual paraffin. The slides are then rehydrated for 2 × 5 min in decreasing
concentrations of ethanol in water, starting at 100% ethanol before moving to 96% and 70% ethanol. Finally,
the slides are placed in water for a period of 5 min. The deparaffinization and rehydration process is critical
to unmask epitopes for immunohistochemical labelling. To further assist the access of antibodies to antigens
in the tissue, antigen retrieval can be performed. Different forms of antigen retrieval methods are described
in the literature. In our case, citrate buffer antigen retrieval worked best. For antigen retrieval, slides are
incubated in 10 mM citrate buffer (1.8 μM citric acid and 8.2 μM sodium citrate tribasic dihydrate in water)
and placed in an RHS-1 Microwave Vacuum Histoprocessor (Milestone Medical). The microwave heats the
solution to 120 °C and maintains this temperature for an additional 1 min. Slides are then removed from the
microwave and allowed to cool down to room temperature. This step is critical to minimize excess
background signal. Slides are washed in PBS for 5 min prior to permeabilization with 0.5% TritonX-100 for