75, 90, 95, 100, 100%) and washed in this solution at 37˚C for
24 hours. The tissue was then rehydrated in PBS and labelled with
primary antibodies for 1 week at 37˚C (mouse anti-TDP43 and 2x
anti-RFP,Table 2). The tissue was washed for 24 hours and
incubated with appropriate secondary antibodies ( Table 2)f o r
another week at 37˚C. The tissue was subsequently washed for
24 hours, dehydrated again in increasing concentrations of 1-
propanol containing 0.3% triethylamine, and mounted in benzyl
alcohol with benzyl benzoate (1:2 ratio) containing 0.3% triethyl-
amine on glass slides with silicone spacers. Imaging was
performed on an Olympus spinning disk confocal microscope at
20x, with 2- mm z-steps. The tissue was stored at 4˚C for
;16 months before imaging, so only the tissue that remained
transparent at this time was used for downstream analyses.
Volumetric analyses were performed using Imaris using the “spots”
feature with region growth (to allow for different-sized spots),
background subtraction, and point spread function elongation
(standard 23 XY). Initial spot diameters were set based on
MrgDCreERT2;Ai14 nuclear size (as labelled by red fluorescent
protein (RFP)). Spot classification was then performed blind by
adjusting the quality threshold to balance detection in superficial
and deep tissue. This step was necessary due to differences in
tissue quality after long-term storage. Any labelled spots in the
adjacent nerve were then deleted (eg, labelled Schwann cells or
debris). Count and volumetric data were then exported for analysis
in R. Data were filtered for very small ( ,5 mm3) and very large
(.2000 mm3) spots to further remove any debris, labelled satellite
glia or doublets within the ganglia. In both cases, these filters were
approximate and did not exclude the possibility that some spots
correspond to either class in the final dataset. The upper limit of the
“small” DRG nuclei size category was defined as the upper bound
of 32 easily identifiable MrgD 1 nuclei (258 mm3). The boundary
between “medium” and “large” bins (400 mm3) was less clearly
defined in the samples and was therefore set as the approximate
midpoint of the volume distribution. A combined size category for
all nuclei greater than 258 mm3 was also examined, and the results
mirrored those of “medium” and “large” bins.
2.7. Gene Ontology
Gene Ontology term analyses were performed on previously
published mouse subtype RNA-seq after SNI (GSE216444 3). In
this study, subtype-specific bulk RNA-seq was performed on 5
transgenic mouse lines through reporter labelling and fluores-
cence activated cell sorting. spliced transcripts alignment to
a reference was used to map reads to the GRCm38 (mm10)
Mouse Genome,14 and Samtools was used to sort, index, and
merge Binary Alignment Map files in line with published reports. 28
Quality control was performed as per Barry et al. 3 Downstream
analyses were performed using DESeq2 on grouped male and
female samples. 31 For differentially expressed genes (false
discovery rate) (FDR , 0.05, LFC .1) (log-fold change), GO
analyses were performed using the Wallenius method using
goSeq (R). In this study, significantly regulated terms related to
2866 A.H. Cooper et al. ·165 (2024) 2863–2876 PAIN®