Cytochemical staining of endocytosed HRP, flat embedding v1

In this protocol, neurons are pulse-chased with horseradish peroxidase (HRP) to induce activity-dependent uptake of HRP into endocytic compartments such as synaptic vesicles and endosomes. HRP is visualised via diaminobenzidine cytochemistry followed by osmium tetroxide, producing a dark brown, electron-dense precipitate within labeled structures. After staining, neurons are flat-embedded in LX112 directly on the plastic culture dish.