IsoB4 Staining of Brainstem Neurons

Staining procedures were modified from previous descriptions [11 (link)]. Briefly, brainstem sections containing the MNTB were selected and incubated in a blocking solution containing (in %) 1.0 goat serum and 0.4 Triton X-100 dissolved in 0.1 M PB for 30 min at room temperature (RT). Brainstem sections were transferred to a primary label solution containing (in %) 1.0 goat serum, 0.3 Triton X-100, and biotinylated IsoB4 (1:500; Vector Labs, Burlingame, CA, USA), incubated for 2 h at RT followed by overnight incubation at 4 °C. After IsoB4 incubation, brainstem sections were washed twice in 0.1 M PB and incubated for 2 h at RT in secondary label solution containing (in %): 0.02 Triton X-100 and Alexa Fluor-conjugated streptavidin (1:500; Thermo Fisher Scientific, Waltham, MA, USA) followed by overnight incubation at 4 °C, washed 4 times in 0.1 M PB and mounted onto glass slides using a mounting medium with DAPI as a counterstain (Fluorogel, Electron Microscopy Sciences, Hatfield, PA, USA).