We purified retinal ganglion cells from postnatal day 5 rats by sequential immunopanning to greater than 99% purity and cultured them in serum-free medium as previously described41. Control and A1 reactive astrocytes were plated on inserts and co-cultured with RGCs for 5–10 days. For quantification of structural synapses, RGCs were fixed and stained with antibodies against the presynaptic marker Bassoon and postsynaptic marker Homer. Synapse number and size were quantified by a custom-written MATLAB program14.
