Quantifying Neuroinflammation in Cln6 Mice

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)].