Protease free bovine serum albumin

Sections (12 μm) were cut from paraffin blocks using a cryostat at −20°C, mounted onto gel coated slides with frosted ends (Electron Microscopy Sciences, Hatfield, PA, USA), air dried for 1 h, stored at −80°C, and then brought to room temperature before further processing. In situ GM-1 preservation was achieved by cold (−20°C) anhydrous acetone (Sigma Aldrich) fixation and permeabilization for 3 min followed by drying for 15 min in coplin jars [Heffer-Lauc et al., 2004 (link); Heffer-Lauc et al., 2007 (link); Petr et al., 2010 (link); Scharwz et al., 1997 (link)]. Individual wells were formed for each section with a pap-pen (Sigma Aldrich). Sections were then blocked with ice cold PBS Plus (10 mg/mL protease-free bovine serum albumin, 5% normal goat serum, 0.01% NaN₃; Jackson ImmunoResearch, West Grove, PA, USA) for 1 h at 4°C, incubated in CtxB-594 diluted in PBS Plus for 6 h at 4°C, rinsed with cold PBS Plus 10·5 min on an orbital shaker, incubated with DAPI (1 mg/mL; Life Technologies, Carlsbad, CA, USA) in PBS for 1 min, and then sequentially rinsed with deionized water and immunofluorescence mounting solution (Electron Microscopy Sciences, Hatfield, PA, USA) before applying fresh mounting medium. Coverslips were placed and sealed with nail varnish. Sections were kept moist throughout the procedure to avoid immunofluorescence artifacts.