Cm1850

Brain tissues were collected from APP/PS1 transgenic mice. Serial sections (10 μm thick) were cut using a cryostat (Leica, CM1850, Buffalo Grove, IL, USA). Slides were rehydrated in a graded series of ethanol and submerged in 3% hydrogen peroxide to eliminate endogenous peroxidase activity. Colocalization of A2M and Aβ was determined using an immunofluorescence staining kit according to the manufacturer’s instructions (MXB Biotechnologies, Fuzhou, Fujian, China). Briefly, frozen sections of mouse brains were treated with 5% BSA for 1 h and then incubated with a rabbit anti-A2M antibody together with a mouse anti-Aβ overnight at 4 °C. After washes, sections were incubated with Alexa Fluor 555- or 488-conjugated secondary antibodies for 1 h at room temperature. After thorough rinsing, stained sections were dehydrated, cleared and mounted with a fluorescent sealing agent. The sections were examined, and images were obtained using a Leica microscope (Leica, CM1850, Buffalo Grove, IL, USA).

At the end of the study period, EAE mice were deeply anesthetized with an i.p. injection of ketamine (10 mg ml⁻¹, Boehringer Ingelheim) plus xylazine (1.17 mg ml⁻¹, Bayer) and transcardially perfused with saline (0.9% NaCl + 5 M EDTA) for 5 min followed by 4% PFA for an additional 5 min. The spinal columns were isolated and post-fixed in 4% PFA at 4 °C overnight. The next day, the spinal cords were extracted, washed in 1× PBS and immersed in a 30% sucrose (in PBS) cryoprotectant solution for at least 72 h at 4 °C. The spinal cords were then embedded in optimum cutting temperature medium in a plastic mould and frozen on 2-methylbutane (Thermo Fisher Scientific) on dry ice. The tissue blocks were cryo-sectioned (30 μm for Cx3cr1-YFP^creERT2R26^tdTomato EAE mice, 10 μm for all of the other EAE experiments) using a cryostat (Leica, CM1850) with a microtome blade (Feather) onto Superfrost Plus slides (Thermo Fisher Scientific). The sections were stored at −80 °C until use.
Human tissue was obtained from the Multiple Sclerosis and Parkinson’s Tissue Bank (Imperial College London) under ethical approval (IRAS reference: 279989). Autopsy flash-frozen brain tissue from one case with secondary progressive MS (44 years old, male) was cryo-sectioned with a 10 μm thickness using a cryostat (CM1850, Leica) with a microtome blade (A35, Feather). The sections were stored at −80 °C until use.

To visualize mitochondrial morphology in targeted cells, mice were crossed with ROSA26^+/SmY mice. To visualize endogenous mt-YFP signal, sciatic nerves were dissected out, embedded in O.C.T. (Tissue-Tek) and were cut longitudinally at a thickness of 7 μm using a cryostat (CM1850, Leica). For cryosections, the skin was cryoprotected in 15% sucrose for 2 h and then in 30% sucrose overnight, embedded in O.C.T. (Tissue-Tek), frozen on dry ice and sectioned with a cryostat (CM1850, Leica). 10 μm frozen sections were directly mounted for imaging. For all analyses performed in the brain, vibratome sections were incubated with an anti-GFP antibody to enhance the endogenous YFP signal. Fluorescent images were acquired using a gSTED super-resolution and confocal microscope with HyD detector (TCS SP 8, Leica), as specified. The circularity of mitochondria in targeted oligodendrocytes within the corpus callosum was measured using a macro of ImageJ. The circularity was calculated with the following formula: 4 *pi*(area/perimeter^2) [57 (link)].