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vonderEmbse et al. Neuroimmunol Neuroinflammation 2020;7:345-59 I http://dx.doi.org/10.20517/2347-8659.2019.29 Page 349

with Harris’ Alum Hematoxylin. After sequential washing in ethyl alcohol and Histo-Clear, slides
were coverslipped with Permount (Fisher Scientific, Fair Lawn, NJ, USA) and cured overnight before
visualization.

Slides were visualized using a Leica DM1000 light microscope at 20× magnification (image resolution at
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1.35 µm/pixel) with a SPOT Idea camera attachment and Advanced Imaging software. Regions of interest
(ROI) were chosen at random along the dentate gyrus with the viewing frame containing as much tissue
as possible and averaged for each of the 3-4 serially-sliced tissue sections per sample for a total of n = 6-8
ROIs/animal. Negative and positive controls were routinely employed to determine immunopositive DAP12
reactivity and to instruct background thresholding to minimize batch effects. Immunopositive DAP12 was
[47]
determined over a manually predetermined background threshold via FIJI analysis, blinded to sample
grouping. Use of a Color Deconvolution plugin using predetermined vectors for DAB and hematoxylin
[Supplementary Figure 1], and the % area DAP12 positive/ROI was determined along the dentate gyrus
hippocampal subregion. Mean % area DAP12+ was averaged from n = 6-8 ROIs (technical replicates) per
animal, for each of the n = 3 animals/exposure/sex/age/strain. All data are represented as the mean % over
control at PND10 ± SEM. Raw, untransformed values for % DAP12 immunopositive staining at each age,
and exposure are listed in Supplementary Figure 2, stratified by sex, strain, and age.

Quantitative real-time polymerase chain reaction
RNA Isolation and cDNA synthesis
Total RNA was purified from no more than 20 mg frozen brain tissue taken from the cortex of the right
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hemisphere of 3xTgAD or WT mice using the miRCURY RNA Isolation Kit - Cell & Plant (Exiqon
Inc., Woburn, MA, USA) and associated Lysis Additive (Exiqon) specific for fatty tissue. Following
homogenization and cell lysis, RNA was purified against a proprietary resin spin column separation matrix,
washed with the associated buffers, and eluted at 50 µL, as per manufacturer’s instructions. Purified RNA
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C
was measured on a NanoDrop One/One Microvolume UV-Vis Spectrophotometer (ThermoFisher
Scientific, Madison, WI, USA) for purity and concentration, which was then adjusted to 5 ng/µL for
subsequent reverse transcription.

For each sample, cDNA was synthesized from 10 ng purified RNA, in duplicate, using the Universal cDNA
synthesis kit (Exiqon) and the following protocol: 60 min at 42 °C, followed by heat-inactivation of the
reverse transcriptase for 5 min at 95 °C. Newly synthesized cDNA was then stored at -20 °C, and thawed
and diluted to 80× in RNase-free water before RT-PCR.

Real-time PCR
MicroRNA relative quantification was performed on an iQ5 Multicolor Real-Time PCR Detection System
thermocycler (Bio-Rad Laboratories, Inc., Hercules, CA, USA), using 96-well PCR plates (VWR, Radnor,
PA, USA). A master mix was prepared for each pre-designed LNA PCR Primer set (Table 1, Exiqon), in
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conjunction with ExiLENT SYBR® Green master mix (Exiqon), for the target primers (miR-124, miR-132,
miR-34a), endogenous reference primer (SNORD110), and UniSp6 RNA Spike-in control primer included
in the kit. Each well consisted of 6 μL master primer mix and 4 μL cDNA template per sample, for a
final well volume of 10 μL per sample, performed in duplicate. Reaction conditions included polymerase
activation/denaturation at 95 °C for 10 min, 40 amplification cycles at 95 °C for 10 sec and 60 °C for 1 min
(1.6 °C/s ramp rate), and a melt curve analysis consisting of a setpoint at 60 °C and endpoint at 95 °C,
incrementally increasing by 0.5 °C with a 10 s dwell time.

[48]
Automatically generated threshold cycle values (C) were evaluated using LinRegPCR quality assessment
t
to account for amplicon and assay efficiency. Fold change in relative miRNA expression compared to
sex-, strain-, and age-matched controls was determined by the Pfaffl method, accounting for differential
amplicon efficiencies calculated by LinRegPCR.