De-identified paraffin-embedded tissue sections from sporadic ALS patient and control cervical spinal cord tissue were provided by the Target ALS Multicenter Postmortem Tissue Core (www.targetals.org). Then, 3 × 5 min washes with Clear-Rite 3 (ThermoFisher, Cat: 6901TS) were used to de-paraffinize the sections. Slides were rehydrated in a series of 5 min EtOH washes (2 × 100%, 1 × 90%, 1 × 70%) and then washed 3× with ddH20. Antigen retrieval was performed by steaming in Epitope Retrieval Solution (IHC World, Cat: IW-1100) for 1 h then washed 3× with PBS. Sections were permeabilized for 10 min with 0.4% Triton X100 in PBS then washed 3× with PBS. Autofluorescence was reduced by incubating with 1× TrueBlack in 70% ethanol (Biotium, Cat: 23007) for 30 s, then washed 3× with large volumes of PBS. Blocking was conducted with 5% Donkey Serum (SigmaAldrich, Cat: D9663) in PBS overnight at 4 °C. All primary antibodies were diluted in 2.5% Donkey Serum in PBS and applied for 24 h at 4 °C. Subsequently, slides were washed 3× with PBS, then incubated in secondary antibodies diluted in 2.5% Donkey Serum in PBS for 1 h at RT. Slides were then washed 3× with PBS before mounting with fluorescent mounting media. Primary antibodies utilized: 5 μg/mL Rabbit anti-IBA1 (Wako, Cat: 013-27691), 4 μg/mL Goat anti-RAGE (R&D, Cat: AF1179), 5 μg/mL Mouse anti-GFAP (BD Biosciences, Cat: 556330), and 5 μg/mL Mouse anti-NeuN (Millipore, Cat: MAB377). Secondary antibodies utilized: Donkey anti-rabbit Alexa Fluor 488 (Invitrogen, Cat: A21206), Donkey anti-mouse Alexa Fluor 488 (Invitrogen, Cat: A21203), Donkey anti-Goat Alexa Fluor 594 (Invitrogen, Cat: A32758), and Donkey anti-Goat Alexa Fluor 647 (Invitrogen, Cat: A32849). All secondary antibodies were used at 1 μg/mL. As above, all experiments included negative controls with omission of primary antibodies.

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