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Page 6 of 9 Klugmann et al. Rare Dis Orphan Drugs J 2023;2:8 https://dx.doi.org/10.20517/rdodj.2023.05

Figure 2. Representative chromogenic detection of AspRS by immunohistochemical labelling with Diaminobenzidine (DAB) in the motor
cortex, hippocampus, brainstem (pons), and cerebellum of the human brain. Scale bar: 100 µm.

Sections are washed 3 times with PBS and then incubated with biotin-conjugated secondary antibodies for 2
h at room temperature (instead of fluorophore-coupled secondary antibodies). Sections are washed 3 times
in PBS and incubated in ABC solution (Vectastain #PK-6105, Goat IgG) for 30 min at room temperature.
Following three washes with PBS, sections are incubated with DAB solution (DAB substrate kit for
peroxidase; Vector Laboratories #SK-4100) as described in the manufacturer’s protocol. DAB-labelled
sections can be imaged with a suitable light microscope or scanned using a slide scanner. In our study, an
Aperio slide scanner (Leica) was used, followed by image processing using the Aperio ImageScope software
(Leica) [Figure 2]. Representative chromogenic images showing AspRS in the cortex, hippocampus,
brainstem, and cerebellum of the human brain using the DAB staining method described here are shown in
Figure 2. An in-depth characterization and discussion of AspRS expression in the human brain has been
described elsewhere .
[12]

TECHNICAL NOTES
Western blotting
The homogenization method used for protein extraction may vary depending on the kind and amount of
tissue. For brain tissue, we found that a combination of sonication and grinding in liquid nitrogen works
best. When grinding using mortar and pestle, make sure that the sample remains covered in liquid nitrogen
to avoid thawing of the tissue. Before the pulverized tissue is transferred into an Eppendorf tube, ensure that
all liquid nitrogen has evaporated from the mortar. Any liquid nitrogen transferred into an enclosed
reaction tube can be hazardous as the tube might explode when warming up. During the subsequent
sonication step, all cells are lysed by liquid cavitation. An advantage of sonication over other protein
extraction methods is that DNA is also sheared during the process and there is no need to treat the sample
with DNase. To avoid protein degradation, it is important to keep the samples on ice during sonication.