Open Access

Diane Bissen, Maximilian Ken Kracht, Franziska Foß, Amparo Acker-Palmer

• Highlights • Enables immunostaining and visualization of epitopes deep within brain slices • Utilizes expansion microscopy to increase imaging resolution • Optimized for brain organotypic slice cultures and tested in acute brain slices • Analysis workflow for protein distribution (surface vs. intracellular pool) using Imaris Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Summary Assessing protein distribution with super-resolution in tissue is often complicated and restrictive. Here, we describe a protocol for immunostaining and expansion microscopy imaging of mouse brain organotypic slice cultures. We detail an Imaris analysis workflow to analyze the surface vs intracellular distribution of AMPA receptors at super-resolution during homeostatic plasticity. We have optimized the protocol for brain organotypic slice culture and tested in acute brain slices. This protocol is suitable to study protein distribution under multiple plasticity paradigms.