S9378

Post-fixed spinal cords were cryo-protected in 15% and 30% sucrose (Sigma, S9378) in 1×PBS. Spinal cords were mounted in cryomolds (Tissue-Tek® Cryomold®, 420572) using compound (Tissue-Tek® O.C.T.™) and rapidly frozen to − 60 °C. Twenty-micrometer coronal sections were produced using a cryostat (Leica, CM1850, − 22 °C) and mounted on slides (VWR, SuperFrost® Plus, 48311-703). Sections were thawed, rehydrated in 1×PBS, blocked for 2 h at RT in blocking solution (0.3% Triton X-100 (Sigma, 93443), 5% normal goat serum (Serotec, 301104, 1×PBS and 0.01% sodium azide (Sigma, S-2002)). Primary antibody (Table 1) was added, and sections were incubated at 4 °C for 24 h. Sections were rinsed in 1×PBS followed by incubation in secondary antibody (Table 1) at RT for 1 h. Sections were incubated at RT for 20 min with nucleic acid stain (Hoechst 33258, Invitrogen™ H3569). Prior to confocal microscopy, the slides were rinsed and mounted using Mowiol (Sigma, 81381) and a cover slip (Marienfeld, 010243).

Mitochondria were extracted from HA and A172 cells through the Mitochondrial extraction kit. Isolated mitochondria were suspended within a solution with 0.25 M sucrose (Sigma, S9378), 1 mM ethylenebis (oxyethylenenitrilo) tetraacetic acid (EGTA), and 5 mM Tris, pH 7.4 at 0 °C. The proton pump rate (PPR) underwent the K₃Fe(CN)₆ pulse method as in our previous study [13 (link)]. Electron transport rates and proton ejection were determined using 557 double-beam spectrophotometers (PerkinElmer, Waltham, MA, USA) and PHM84 fast-responding pH electrode system (Radiometer Medical, Copenhagen, Denmark).

Eyes were harvested from mice and the retina and lens were isolated as described above. The retina and lens were fixed in 4% paraformaldehyde at 4 °C for 1 h. The tissues were washed with 1X PBS, incubated overnight in 15% sucrose solution (Sigma-Aldrich S9378) at 4 °C and then transferred to 30% sucrose at 4 °C for an additional night. The tissue was then embedded in OCT compound (Sakura 4583) on dry ice and cryosectioned into 20 μm sections. Cryosections were washed with 1X PBS-T (1X PBS + 0.1% Triton-X 100), 3 times for 10 min each, and then blocked in 2% normal goat (Sigma-Aldrich G9023) or donkey serum (Sigma-Aldrich D9663) for 1 h at room temperature. Next, the tissue was stained with primary antibodies (diluted in blocking solution) overnight at 4 °C in a humid chamber. Then, the tissue was washed with 1X PBS for 3 times, 10 min each at room temperature and stained with secondary antibodies (in blocking solution) for 1 h at room temperature in a dark humid chamber. The tissue was again washed with 1X PBS 3 times for 10 min and a coverslip was mounted over the tissue with Fluoromount-G (Southern Biotech, #0100–01). Primary and secondary antibody concentrations are listed in Additional file 2.

Freshly dissected endocrine glands collected from mice were immediately fixed in ice‐cold 2% paraformaldehyde solution for 4 h. Next, the fixed gland tissues were cryoprotected by overnight incubation with 20% sucrose (Sigma‐Aldrich, S9378) and 2% polyvinylpyrrolidone (PVP, Sigma‐Aldrich, PVP360) solution overnight at 4°C. Then, the tissues were embedded and frozen in 8% gelatin (Sigma‐Aldrich, G2625) solution supplemented with 20% sucrose and 2% PVP. Thick sections (100–150 μm) were generated by low‐profile blades (Leica, 14035838382) on a Leica CM3050 cryostat. All the endocrine glands were sectioned longitudinally, and sections from the middle/centre of the glands were used, while the sections at the periphery were excluded. Sections were then air‐dried for 5 h, frozen and stored until used for immunostainings.

HEK293T cells expressing WT PINK1, or CRISPR edited to express PINK1^G411A or PINK1^G411S were treated with 10 µM CCCP for the time indicated. Cells were harvested and subjected to crude mitochondrial isolation as described previously [20 (link),31 (link)]. Briefly, crude mitochondria samples were prepared by sonication of cells resuspended in mitochondrial isolation buffer (50 mM Tris-HCl, pH 7.5, 70 mM sucrose [Sigma-Aldrich, S9378], 210 mM sorbitol [Sigma-Aldrich, S1876], 1 mM EDTA, 1 mM EGTA, 50 mM NaF, 5 mM sodium pyrophosphate, 10 mM sodium 2-glycerophosphate [Calbiochem, 35,675], 1 mM AEBSF [Sigma-Aldrich,76,307], 10 mM PR-619 [Selleck Chemicals, S7130], 1 mM benzamidine [Sigma-Aldrich, B6506], 1 mg/ml leupeptin [Sigma-Aldrich, L2023] and aprotinin [Roche, 10,236,624,001]) plus 100 mM chloroacetamide (Sigma-Aldrich, C0267) and mitochondria were then isolated by differential centrifugation before lysis. Mitochondrial extracts were sonicated, clarified by centrifugation, and protein concentrations determined by the Bradford assay (Bio-Rad, 5,000,006). Quantitative mass spectrometry was performed on mitochondrial extracts (3 µg) using PRKN target-Parallel Reaction Monitoring (Pt-PRM) platform using an optimized series of heavy reference peptides for PRM analysis of Ub chain linkages, and p-S65-Ub [52 (link)] and data were collected on an Orbitrap Fusion Lumos instrument.

Embryos were subjected to a sucrose gradient (5–20%) followed by embedding in a 1:1 mixture of Tissue-Tek O.C.T. (Sakura, 4583) and 20% sucrose (Sigma-Aldrich, S9378), and frozen in a bath of 2-methylbutane chilled with liquid nitrogen. Samples were stored at −80 °C or cryo-sectioned into 8 µm slices transferred to polysine-coated slides. Sections were fixed in 4% PFA at RT for 10 min. After washing with PBS, samples were incubated in solution containing 10% FBS, 0.1% Triton X-100 in PBS at RT for 1.5 h. Next, they were incubated with primary antibodies (Supplementary Data 10) diluted in 1% FBS, 0.1% Triton X-100 in PBS at 4 °C overnight. The next day, samples were washed 3x with 0.1% Triton X-100 in PBS for 15 min total and incubated with secondary antibodies (Supplementary Data 11) and 5 μg/ml Hoechst 33258 diluted in 1% FBS, 0.1% Triton X-100 in PBS at RT for 1 h. After another round of washing, sections were covered with mounting medium and a cover slip and stored at 4 °C.

Mice were killed by carbon dioxide (CO₂) inhalation and perfusion fixed with 10% buffered formalin via the left ventricle for 5 min. Then we dissected the ankles with Achilles tendons and fixed the specimens in 10% buffered formalin for 24 h, decalcified in 10% Ethylenediaminetetraacetic acid (EDTA, VWR, 0105) (pH 7.4) for 14 days, and embedded in paraffin, Optimal Cutting Temperature Compound (O.C.T. compound, VWR, 25608-930,) for 3 days and embedded in matrix containing 8% Gelatin (Sigma-Aldrich, G1890), 20% sucrose (Sigma-Aldrich, S9378), and 2% Polyvinylpyrrolidone (Sigma-Aldrich, PVP40) at −80 °C adjusted from previous protocol^{68 (link)}. The majority of analyses were in paraffin-embedded specimens, while detection of Nestin, CD31, and Emcn was more optimal in frozen specimens.