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

Autofluorescence Eliminator reagent (Millipore #2160) works well on human tissue.

When choosing the most appropriate antibody, it is important to consider the specific advantages and
[13]
disadvantages of polyclonal versus monoclonal antibodies . Polyclonal antibodies are produced by
injecting an animal with the desired antigen to elicit an immune response and produce antibodies against
the antigen. Polyclonal antibodies can recognize multiple epitopes of the antigen, which often results in a
higher overall affinity and sensitivity to the target protein. The same multi-epitope specificity, however, can
result in cross-reactivity with other proteins leading to increased unspecific binding and background
staining. In contrast, monoclonal antibodies are produced in vitro by a single B cell clone and recognize
only one specific epitope. While this might result in lower overall sensitivity, monoclonal antibodies often
show higher specificity with reduced cross-reactivity and, consequently, less background signal. If working
with brain tissue from patients affected by a known ARS mutation, a monoclonal antibody that recognizes
an epitope outside the mutated amino acid sequence should be used. Alternatively, a polyclonal antibody
can be used to ensure multiple epitopes are recognized.

Studies involving antibody-based methods should always include appropriate controls for the specificity of
antibodies, such as secondary-only stainings. A comprehensive guideline for the validation of antibodies for
use in immunohistochemistry has been developed by Howat et al. . Determination of antibody specificity
[14]
is crucial in studies on human tissue when genetic manipulations such as overexpression or knockdown via
RNA interference of the protein of interest cannot be performed to confirm the validity of an antibody. If
positive or negative human control tissue with confirmed up- or downregulated target protein is available,
this can be used for establishing antibody specificity. Western blotting can give a first indication if an
antibody is specific or not (see above); however, it does not always guarantee antibody specificity in
immunohistochemistry applications where the tissue has undergone different processing steps, including
fixation and antigen retrieval. A good indication for the specificity of an antibody can also be obtained by
ectopically expressing the human protein of interest in a cell culture model such as HEK293 cells with
subsequent immunocytochemistry. Finally, peptide blocking, as described above, can be employed to rule
out non-specific binding in immunohistochemistry. If the above controls are not possible or yield
inconclusive results, orthogonal methods such as in situ hybridization of the corresponding mRNA should
be applied to support the antibody-based data.

DECLARATIONS
Acknowledgements
The authors thank the New South Wales Brain Bank for providing the human tissue samples.

Authors’ contributions
Led the project and manuscript preparation: Klugmann M, Fröhlich D
Conducted the research: Klugmann M, Suchowerska AK, Housley GD, Fröhlich D
All authors contributed to and approved the final version of the manuscript.

Availability of data and materials
Not applicable.

Financial support and sponsorship
This work was supported by the European Leukodystrophy Association (ELA 2018-014I2) and the
Australian Government Medical Research Future Fund (Leukodystrophy Flagship - Massimo’s Mission;
MRFF-ARLKO).