Live5

Thin sections of paraffin-embedded tissues (brain and pancreas) were de-paraffinized, blocked in blocking solution (10% goat serum+5% BSA+0.5% triton X-100) and then incubated with primary antibodies at 4°C overnight. In the immunofluorescence measurements, a combination of anti-amylin (SC-377530; Santa Cruz biotech; TX, raised in mouse) and anti-4-HNE (ab46545; Abcam; MA, raised in rabbit), anti-MDA (ab94671; Abcam; MA, raised in rabbit), or anti-IL-1β (ab9722; Abcam; MA, raised in rabbit) primary antibodies were used. After washing, sections were incubated with Alexa Fluor 488 conjugated anti-mouse IgG (A11029; Invitrogen; NY) and Texas red conjugated anti-rabbit IgG (SC-2780; Santa Cruz biotech; TX) secondary antibodies. The sections were then stained with DAPI (ab 104139, Abcam; MA) and imaged with a laser-scanning confocal microscope (Live5; Zeiss; Germany). Immunofluorescence measurements were also done on fixed neurons incubated with anti-IL-1β primary and Alexa Fluor 488 conjugated secondary antibodies. Immunofluorescence staining for amylin was verified with a second anti-amylin antibody (T-4157; Bachem-Peninsula Laboratories; San Carlos; CA, raised in rabbit; Supplementary Figure 1). Tissue autofluorescence and elastin autofluorescence were blocked with 1% Sudan black (Supplementary Figure 2).

Primary cultured neurons were loaded with the fluorescent probe 4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a, 4a-diaza-s-indacene-3-unde canoic acid (C₁₁-BODIPY^581/591; D3861; Invitrogen; OR) and imaged with a fluorescence microscope (Live5; Zeiss; Germany). Upon peroxidation, the fluorescence emission peak of C₁₁-BODIPY^581/591 shifts from 590 nm (red) to 510 nm (green). Thus, lipid peroxidation was measured as the ratio between the average fluorescence intensity in the green and red channels. A previous study [27 (link)] reported the tendency of this fluorescent dye to overestimate lipid peroxidation and to inhibit oxidative damage, but confirmed its capability to detect oxidative stress conditions at the membrane level.

For calcium imaging in primary neural cells, ~50 third instar Tg larval brains expressing Mito-GFP, Mito-GFP+UAS-dMiro-WT, Mito-GFP+UAS-dMiro-S66A, or Mito-GFP+dMiro RNAi driven by elav-GAL4 were collected. Primary neural cells were prepared according to published procedures (Egger et al., 2013 ). To monitor mitochondrial or cytosolic Ca²⁺ levels, primary neural cells were loaded with 1 μM Rhod-2 AM or Fluo-3 AM (Molecular Probes), respectively, in physiological salt solution [pH7.4] (150 mM NaCl, 4 mM KCl, 1 mM MgCl₂, 5.6 mM glucose, 5 mM HEPES) for 30 min at 37 °C. Primary neural cells were treated with 1 μM Thapsigargin (Calbiochem) for intracellular Ca²⁺ perturbation. Calcium imaging was carried out using an inverted confocal microscope (LSM 510 META and LIVE 5, Carl Zeiss) with a 40× objective.
For measuring mitochondrial Ca²⁺ in mammalian cells, HeLa cells were transfected with control pCDNA3.0 vector or Myc-hMiro1, Myc-hMiro2, Myc-dMiro-WT, Myc-dMiro-S66E, or Myc-dMiro-S66A plasmids cloned in pCDNA3.0. After transfection, HeLa cells were loaded with Rhod-2 AM (10 μM in DMEM media, Molecular Probe) for 30 min at room temperature. Cells were then washed with DMEM for 30 min. Single cell fluorescence was excited at 545 nm and images of the emitted fluorescence obtained on a Leica TCS SP5 AOBS confocal microscope were processed with LAS AF (Leica).