Ab181243

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)].

For Western blot analysis, the following antibodies were used in a 1:1000 dilution: anti-β (ab14730, Abcam), anti-γ (PA5-29975, ThermoFisher), anti-b (ab117991, Abcam), anti-OSCP (ab110276, Abcam), anti-f (ab200715, Abcam), anti-g (ab126181, Abcam), anti-e (ab122241, Abcam), anti-c (ab181243, Abcam), anti-a (ab192423, Abcam), anti-citrate synthase (ab96600, Abcam), anti-vinculin (V4505, Sigma), anti-prohibitin (MS-261-P1, NeoMarkers), anti-GAPDH (2118, Cell Signaling), anti-CyPD (ab110324, Abcam), anti-ANT2 (14671, Cell Signaling), anti-ANT3 (PA5-35113, ThermoFischer), anti-OXPHOS (ab110411, Abcam), anti-HKII (sc130358, Santa Cruz), anti-UQCRC1 (sc65238, Santa Cruz), anti-Grim19 (sc271013, Santa Cruz), and anti-SDHA (sc166947, Santa Cruz). Statistical comparison of data was assessed with the two-sided Student’s t-test.

Activation of the mitochondria-dependent cell death pathway was assessed by detection of cytochrome c release from mitochondria. This was done by immunofluorescence analysis using antibodies against cytochrome c and ATP synthase subunit C, which serves as a mitochondrial marker. Briefly, NRVMs were exposed to 1% CSE in serum-free DMEM for 1 h. After PBS washing, cells were fixed with 4% paraformaldehyde (10°C, 10 min) and permeabilized with 0.2% Triton-X 100 (room temperature, 1 min). After blocking with Blocking One Hist (Nacalai Tesque, Kyoto, Japan) for 10 min, NRVMs were treated with primary antibodies (anti-cytochrome c: mouse, 1/200, ab110325, and anti-ATP synthase subunit c: rabbit, 1/200, ab181243, abcam, Cambridge, UK) and incubated overnight at 4 °C. Secondary antibodies (Anti-mouse IgG Alexa fluor 488: 1/200, and Anti-rabbit IgG Alexa fluor 594, 1/200, Invitrogen) were then added, and nuclei were further stained with Hoechst 33342 (Invitrogen). Fluorescence images were acquired using LSM900 confocal microscopy and ZEN3 imaging software (Carl Zwiss). Colocalization of cytochrome C and ATP synthase subunit C in the myocytes treated with 1% CSE and Tyrode were analyzed by Pearson’s correlation using Image J.

Cells were seeded on coverslips, fixed with a 4% paraformaldehyde (PFA) solution, blocked, and incubated with primary antibodies overnight at 4 °C. The primary antibodies were anti-SCMAS (1/200) (ab181243; Abcam), anti-HSP60 (1/500) (ab46798; Abcam) and anti-LAMP1 (1/100) (1D4B; Developmental Studies Hybridoma Bank). They were then incubated for 1 h with fluorescent secondary antibodies (1/500) Alexa Fluor 488 anti-rabbit (A11008; Thermo Fisher), and Alexa Fluor anti-rat 647 (A-21247; Thermo Fisher). DAPI (4′,6-diamidino-2-phenylindole) was used for nuclei visualization. Coverslips were mounted in ProLong Gold antifade reagent. Negative controls were performed with either no primary or no secondary antibodies. No staining was detected in any case. Images were acquired on an Operetta CLS high-content imaging system (PerkinElmer) using ×63 (1.15 numerical aperture) objective. Images were acquired at the same exposure times in the same imaging session. Image quantification was performed after appropriate thresholding using the ImageJ software (NIH). The percentage of colocalization was calculated using the JAva COnstraint Programming (JACoP) plugin, specifically Manders’ Overlap Coefficient, in single Z-stack sections.

Immunoblotting was performed with anti- C-subunit of ATP synthase (SCMAs) (1/1000) (ab181243; Abcam), anti-VDAC (1/666) (PC548; Calbiochem), anti-heat-shock protein-60 (HSP60) (1/666) (ab46798; Abcam), anti-PINK1 (1/500) (sc-33796; Santa Cruz Biotechnology), anti-NDUFS1 (1/500) (sc-50132; Santa Cruz Biotechnology), anti-CDK5 (1/500) (sc-6247; Santa Cruz Biotechnology), anti-PFKFB3 (1/500) (H00005209-M08; Novus Biologicals), anti-p25/35 (1/666) (2680; Cell Signalling), anti-caspase-3 (1/2000) (9661S; Cell Signalling), anti-CLN7 (1/500) (donated by Dr. Stephan Storch), anti-Parkin (1/100) (sc-32282; Santa Cruz Biotechnology), anti-LC3B (1/1000) (2775; Cell Signaling), anti-GFAP (1/500) (G6171; Sigma), anti-β-Tubulin III (1/500) (ab18207; Abcam) and anti-β-Actin (1/30,000) (A5441; Sigma).

The following antibodies were used: anti APTase Subunit C (ab181243, Abcam, Cambridge, MA, USA); anti-PHB (gift from Dr Reusch, Michigan State University); Alexa Fluor_ 680 goat antimouse IgG (catalog number A21057, Invitrogen, Carlsbad, CA, USA); Alexa Fluor_ 750 goat anti-rabbit IgG (catalog number A21039, Invitrogen) and anti-ATPase immunocapture antibody (ab1099867, Abcam, Cambridge, UK).