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Klugmann et al. Rare Dis Orphan Drugs J 2023;2:8 https://dx.doi.org/10.20517/rdodj.2023.05 Page 7 of 9

Choosing the right housekeeping protein is critical for normalization of protein levels, as some
housekeepers might be regulated in certain disease conditions or tissues. Normalization against two
different housekeeping proteins should yield comparable results and is a good control for the validity of the
housekeeper.

Unspecific binding of an antibody - the binding of an antibody to non-target antigens, is easier to identify in
Western blotting compared to immunohistochemistry as the molecular weight at which the protein of
interest is detected is a strong indication of antibody specificity. Detection of multiple bands on a Western
blot can indicate non-specific binding, which needs to be considered if the same antibody were to be used in
immunohistochemistry. If uncertain of antibody specificity, peptide blocking should be performed to
confirm the validity of the primary antibody. For this, the antibody is pre-absorbed with a blocking peptide
corresponding to the epitope of the antibody, which will prevent the binding of the target antigen and
consequently reduce signal intensity compared to non-blocked antibodies.

Quantitative real-time PCR
For RNA work, it is essential to keep the workplace and reagents free of RNases to avoid degradation of the
RNA. It is recommended to perform all RNA work in a laminar flow hood.

Similar to protein extraction, tissue homogenization for RNA preparation is performed in two steps.
Following liquid nitrogen grinding, cells are lysed using a pellet pestle homogenizer instead of a probe
sonicator to avoid shearing of the RNA.

In our study, off-the-shelf TaqMan assays worked best as they provide species-specificity and low unspecific
binding. Selection of the appropriate TaqMan assay should take into account that the primers are exon-
exon junction spanning to avoid detection of genomic DNA. If analyzing human brain tissue from a carrier
of a known ARS mutation, it is essential to select primers and TaqMan probes that anneal to cDNA
sequences unaffected by the respective mutation. The specificity of qRT-PCR primer pairs or TaqMan
assays should always be validated through sequencing of the amplicon.

Immunohistochemistry
Deparaffinization and rehydration of paraffin-embedded sections are critical to unmask epitopes for
immunohistochemical labelling. For some antibodies, it is necessary to perform additional antigen retrieval
to further assist the access of the antibody to the antigen. Which antigen retrieval method should be
employed depends on the antibody used, and it is worthwhile testing them before the study commences. In
our study, citrate antigen retrieval yielded the best results.

To avoid unspecific antibody binding, blocking should be performed in the serum of the host species of the
secondary antibody. If this is not possible, we generally have had good experiences using normal goat
serum.

For immunofluorescent labelling of antigens, we achieved the best results employing secondary antibodies
coupled with Alexa fluorophores.

Human tissue samples inherently give rise to a lot of background signals, as there is no perfusion of tissue
prior to fixation, resulting in autofluorescence. There are numerous contributing factors including collagen
or elastin, which often illuminate blood vessels in the tissue. Historically, Sudan Black has been used to
reduce autofluorescence; however, this also dampens the true fluorescent signal. We have found that the