Alexa fluor 555 donkey anti rabbit

Spinal cord immunohistochemistry was performed as previously described (Smith et al., 2017 (link)). In brief, L3‐L6 segments were sectioned at 50 µm on a cryostat (−20°C) and free‐floating sections were collected and stored in PBS until staining. These were then washed in PBS (3 × 10 min) and incubated for 1 h in blocking solution (0.2% Triton X‐100, PBS, NaCl and 10% normal donkey serum). The free‐floating sections were then incubated for 48 h in primary antibodies diluted in blocking solution, washed and then incubated in secondary antibodies for 2 h, also in blocking solution. Primary antibodies: goat anti‐vesicular acetylcholine transporter (anti‐VAChT, Millipore Cat# ABN100, RRID:AB_2630394, 1:1000), mouse anti‐K_V2.1 (UC Davis/NIH NeuroMab Facility Cat# 73–014, RRID:AB_10672253, 1:200) and rabbit anti‐SK3 (Millipore Cat# AB5350‐200UL, RRID:AB_91797, 1:200). Secondary antibodies at 1:200: Alexa Fluor^® 555 donkey anti‐mouse (Thermo Fisher Scientific Cat# A‐31570, RRID:AB_2536180), Alexa Fluor^® 488 donkey anti‐goat (Jackson ImmunoResearch Labs Cat# 705–546–147, RRID:AB_2340430) and Alexa Fluor^® 555 donkey anti‐rabbit 555 nm (AB_2563181). Finally, sections were mounted on glass slides with Mowoil 4‐88 (Carl Roth GmbH & Co. Kg).