Spine maturation is reflected by decreased spine length and increased size of spine heads leading to a mushroom-like appearance. The following protocol is used to study the involvement of ephrinB reverse signaling in this process. Hippocampal neurons from embryonic rat (E18-19) were isolated and cultured for 14 days. In order to visualize the protrusion the neurons were transfected with Yellow Fluorescent Protein (YFP) 2 days prior to stimulation (12DIV) using Calcium Phosphate method. Activation of ephrinB ligands by soluble EphB receptor ectodomains fused to Fc portions of human IgG (EphB2-Fc) promoted spine maturation in these neurons (14DIV). Spine maturation was analyzed by comparing the average spine length and spine head area of stimulated neurons to that of Fc-stimulated control neurons.