Murine tissue staining

Mice were anesthetized with Ketamine and Xylazine and then underwent rapid cardiac perfusion with PBS, followed by 4% paraformaldehyde (PFA). The spinal column was rapidly removed, and the lumbar spinal cord was retrieved. Lumbar spinal cords were washed in PBS and then drop-fixed in 4% PFA for an additional 1 h at 4 °C. Gastrocnemius tissue was collected and drop-fixed 16 h in 1% PFA at 4 °C. After fixation, tissue was incubated with 15% sucrose in PBS for 24 h followed by incubation with 30% sucrose in PBS for an additional 24 h. Cyroprotected tissue was frozen in optimal cutting temperature (OCT) compound (FisherScientific, Cat: 23-730-571) and kept at − 80 °C until sectioning. Furthermore, 8-μm-thick serial sections were collected on Superfrost PLUS slides (FisherScientific, Cat: 22-037-246) using a Microm cryostat (ThermoFisher, Model: HM550). Sections were washed 3× with PBS for 5 min and then permeabilized with 0.2% Triton-X 100 in PBS for 10 min and washed 3× with PBS. Blocking was conducted with Serum-Free blocking buffer (Dako, Cat: X090930-2) for 1 h at RT. All primary antibodies were diluted in Antibody Diluent (Dako, Cat: S3022) and applied overnight at 4 °C. Subsequently, slides were washed 3× with PBS, then incubated with secondary antibodies diluted in Antibody Diluent for 1 h at RT. For slides containing DAPI, slides were washed and placed in a 1 μg/mL DAPI (Invitrogen, Cat: D1306) solution for 5 min at RT and washed 3× with PBS before mounting with fluorescent mounting media (Dako, Cat: S302380-2). Primary antibodies used: 1 μg/mL Mouse anti-RAGE (Millipore, Cat: MAB5328), 0.5 μg/mL Rat anti-CD11B (M1/70) (Invitrogen, Cat:14-0112-82), 0.25 μg/mL Rat anti-GFAP (2.2B10) (Invitrogen, Cat: 13-0300), 5 μg/mL Rat anti-CLEC7A (Invivogen, Cat: mabg-mdect), 1 μg/mL Mouse anti-AMIGO2 (G-7) (SantaCruz, Cat: sc-373699), 5 μg/mL Mouse anti-NeuN (Millipore, Cat: MAB377), 5 μg/mL Rat anti-CD68 (Abcam, Cat:ab53444), 5 μg/mL Rat anti-F4/80 (Abcam, Cat: ab6640), and 3 μg/mL Chicken anti-MAP2 (Abcam, Cat:ab5392). Secondary antibodies utilized: Donkey anti-Rat Alexa Fluor 488 (Invitrogen, Cat: A-21208), Donkey anti-Mouse Alexa Fluor 546 (Invitrogen, Cat: A10036), Donkey anti-Rat Alexa Fluor 594 (Invitrogen, Cat: A-21209), Donkey anti-Mouse Alexa Fluor 488 (Invitrogen, Cat: A21203), and Donkey anti-Chicken Alexa Fluor 647 (Jackson ImmunoResearch, Cat: 703-605-155). All secondary antibodies were used at 1 μg/mL. All experiments included negative controls by omission of primary antibody.