Immunohistochemical Analysis of Spinal Cord Tissue

Free-floating sections were essentially prepared as described before [21 (link)]. Mice were transcardially perfused with 4% paraformaldehyde (PFA). Subsequently, the spinal cord was removed and post-fixed in 4% PFA for 2 h at RT. After embedding in 5% agarose, 40 μm thick, free-floating sections were cut on a Leica VT1000 S microtome and collected in 0.1 M phosphate buffer pH 7.4. After incubation for 2 h at RT with blocking solution [10% donkey serum, 0.3% Triton X-100 and 0.1% Tween-20 in Tris-buffered saline (TBS)], the sections were incubated for 72 h at 4 °C with primary antibodies in 1:10 diluted blocking solution (1% donkey serum, 0.03% Triton X-100 and 0.01% Tween-20 in TBS). After washing three times for 10 min in TBS-Tween (TBS-T) at RT, sections were incubated for 2 h at RT with the appropriately fluorophore-conjugated secondary antibodies, washed again in TBS-T and finally mounted with Fluor Save (Merck-Millipore). The following primary antibodies were used: polyclonal goat anti-ChAT (MAB144P, Millipore; 1:1000) and polyclonal rabbit anti-TDP43 (10782–2-AP, Proteintech; 1:1000).