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18 reviewsSUMMARYNeuronal cell cultures are highly sensitive to their microenvironment, particularlythe choice of coating substrate. Here, we present a protocol to isolate extracellular matrix (ECM) from mouse brain tissue, providing a native coating materialthat closely mimics in vivo conditions. We detail decellularization steps, guidelines for measuring ECM quality, and instructions for co-culturing neuronal cellson the resulting substrate. By promoting improved neuronal survival, growth,and differentiation, this protocol has broad implications for in vitro neurobiological research and downstream applications.