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Thin-section electron microscopy to visualize APEX-DAB labeled REEP1 vesicles

YS Yoko Shibata
EM Emily E Mazur
BP Buyan Pan
JP Joao A Paulo
SG Steven P Gygi
SC Suyog Chavan
LV L Sebastian Alexis Valerio
JZ Jiuchun Zhang
TR Tom A Rapoport
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U2OS cells stably expressing REEP1-APEX were fixed for 1 h in PBS containing 2% paraformaldehyde/1% glutaraldehyde, quenched with 20 mM glycine for 20 min, and then washed 5x in 100 mM sodium cacodylate buffer. Samples were then incubated with 0.5 mg/ml diaminobenzidine (Millipore #D8001)/0.1 M HCl and 10 mM H2O2 (Millipore #H1009) for 1 h on ice, and washed 5x in sodium cacodylate buffer. Samples were then fixed and stained with 1% OsO4 reduced with 1.5% ferrocyanide, dehydrated sequentially in 20%, 50%, 70%, 90%, and 100% (vol/vol) ethanol on ice, and infiltrated and embedded in Epon resin. Ultrathin sections (~70 nm) were cut on a Reichert Ultracut-S microtome and mounted onto copper grids. Images were acquired on a JEOL 1200X transmission electron microscope equipped with an AMT 2k CCD camera using AMT acquisition software.

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