Mca1957

Three brain sections per animal were selected to contain the ventral posteromedial (VPM)/ventral posterolateral (VPL) nuclei of the thalamus and the somatosensory barrel field (S1BF) cortex. Immunohistochemistry was performed as previously described^{66 (link)}. Briefly, free floating brain sections were incubated in 1% hydrogen peroxide in Tris-buffered saline (TBS) for 20 min to block endogenous peroxidase activity followed by 3 washes in TBS. Sections were then blocked in 15% goat serum-containing TBS-T (TBS with 0.3% Triton X-100) for 30 min followed by primary antibody solution overnight at 4 °C. Primary antibodies included: anti-CD68 (BioRad AbD Serotec, MCA1957; 1:2,000), anti-GFAP (Dako, Z0334; 1:8,000), and anti-ATP synthase C (Abcam, ab181243, 1:1,000) diluted in TBS-T+ 10% goat serum. Secondary antibodies included: biotinylated anti-rat and anti-rabbit IgGs (Vector Labs, BA-9400, BA-1000; 1:2000) diluted in TBS-T+ 10% goat serum. An ABC amplification kit (Vector Labs) was used for 2 h followed by 0.05% DAB solution until visible reaction had occurred.

Wild-type and Cln6^nclf mice were CO₂ euthanized, perfused with PBS, and tissue fixed with 4% PFA. Fixed brains were sectioned on a vibratome at 50 μm (Leica VT10008) and processed with standard immunofluorescence and DAB staining protocols as previously described [44 (link)]. Primary antibodies included anti-CD68 (AbD Serotec, MCA1957; 1:250), anti-GFAP (Dako, Z0334; 1:250), and anti-ATP synthase subunit C (Abcam, ab181243, 1:500). The subunit C experiments were also counterstained with methyl green. Secondary antibodies included anti-rat and anti-rabbit biotinylated (Vector Labs, BA-9400; 1:2000) and Alexa-Fluor fluorescent secondaries (1:1500). Sections were imaged in the VPM/VPL of the thalamus and layers 2/3 of the somatosensory cortex and analyzed using a Nikon 90i microscope with NIS-Elements Advanced Research software (v4.20). For autofluorescent storage material, cells were scored positive for accumulation of storage material when more than three autofluorescent puncta were aggregated around the nucleus. Mitochondrial ATP synthase subunit C, GFAP, and CD68 immunoreactivity was quantified using a threshold analysis in NIS-Elements Advanced Research software, with the subunit C analyzed with the methyl green counterstain excluded from analysis (v4.20) as previously described [44 (link)].

Mice were transcardially perfused with 10 ml of ice-cold PBS followed by 10 ml of ice-cold 4% paraformaldehyde (PFA) in PBS (Roti-Histofix, Roth). Brains were isolated and post-fixed in 4% PFA for 24 h, followed by incubation in 30% sucrose (in PBS, pH 7.5) for a further 48 h. Frozen brains were cut into 25 μm thick coronal sections on a sliding microtome (SM2000R, Leica Biosystems, Wetzlar, Germany) and collected in 15% glycerol. Sections were blocked in 5% normal goat serum (NGS) in PBS with 0.5% Triton X-100. Afterward slices incubated overnight at 4°C with antibodies diluted in PBS containing 1% NGS against the following: anti-Aβ (mouse, 1:1,000, Covance, 6E10), anti-Iba1 (rabbit, 1:1,000, WAKO, 019-19741), anti-CD68 (rat, 1:500, BioRad, MCA1957), anti-GFAP (rabbit, 1:3,000, DAKO, Z033401-2), anti-C3 (rat, 1:200, abcam, ab11862), anti-PDGFRα (rabbit, 1:400, Cell Signaling, 3174), and anti-MBP (rabbit, 1:1000, abcam, ab40390). Corresponding secondary antibodies conjugated to Alexa 488 or 555 (1:1,000) were used. Sections were counterstained with DAPI (Sigma, D9542, 1:10,000) and mounted with a fluorescence mounting medium (DAKO, S3023).