Z033401 2

Formalin‐fixed, paraffin‐embedded tissue blocks from the motor cortex and spinal cord were sectioned at 10 μm and immunostained with antibodies against phospho‐TDP‐43 (S409/410) (Cosmo Bio Co, TIP‐PTD‐M01, 1:80,000), p62 (BD Biosciences, 610833, mouse, 1:250), GFAP (Agilent, Z033401‐2, rabbit, 1:500), Iba1 (Abcam, ab5076, goat, 1:500) and SOD1 (Merck, 574597, sheep, 1:200) and counterstained with 0.5% cresyl violet as previously described [15 (link)]. Immunofluorescence staining was carried out on 10‐μm‐thick sections. Following microwave antigen retrieval (0.01 M citrate buffer, pH 6.0), formaldehyde quenching was carried out using 0.1% sodium borohydride, followed by protein blocking. The sections were then incubated with a primary antibody cocktail consisting of p62 (BD Biosciences, 610833, mouse, 1:250), GFAP (Agilent, Z033401‐2, rabbit, 1:500) and SOD1 (Merck, 574597, sheep, 1:200) antibodies at 4°C overnight. The sections were then visualised with Alexa Fluor 568 donkey anti‐mouse (Thermo Fisher, A10037) and Alexa Fluor 647 donkey anti‐rabbit (Thermo Fisher A‐31573), followed by further blocking with rabbit serum and visualised with AlexaFluor 488 rabbit anti‐sheep (Abcam, 150181). Subsequently, autofluorescence was quenched by Sudan black, and sections were counterstained with DAPI.

Mice were transcardially perfused with 10 ml of ice-cold PBS followed by 10 ml of ice-cold 4% paraformaldehyde (PFA) in PBS (Roti-Histofix, Roth). Brains were isolated and post-fixed in 4% PFA for 24 h, followed by incubation in 30% sucrose (in PBS, pH 7.5) for a further 48 h. Frozen brains were cut into 25 μm thick coronal sections on a sliding microtome (SM2000R, Leica Biosystems, Wetzlar, Germany) and collected in 15% glycerol. Sections were blocked in 5% normal goat serum (NGS) in PBS with 0.5% Triton X-100. Afterward slices incubated overnight at 4°C with antibodies diluted in PBS containing 1% NGS against the following: anti-Aβ (mouse, 1:1,000, Covance, 6E10), anti-Iba1 (rabbit, 1:1,000, WAKO, 019-19741), anti-CD68 (rat, 1:500, BioRad, MCA1957), anti-GFAP (rabbit, 1:3,000, DAKO, Z033401-2), anti-C3 (rat, 1:200, abcam, ab11862), anti-PDGFRα (rabbit, 1:400, Cell Signaling, 3174), and anti-MBP (rabbit, 1:1000, abcam, ab40390). Corresponding secondary antibodies conjugated to Alexa 488 or 555 (1:1,000) were used. Sections were counterstained with DAPI (Sigma, D9542, 1:10,000) and mounted with a fluorescence mounting medium (DAKO, S3023).