Immunofluorescence in 2D and 3D Co-cultures

Immunofluorescence experiments were performed according to Colnaghi et al., 2019 [33 (link)]. Briefly, 2D and 3D co-cultures were fixed in 4% paraformaldehyde (PFA) and a 2% sucrose solution for 30 min, followed by permeabilization with pH 7.4 phosphate-buffered saline (PBS) containing 0.5% Triton X-100 for 1 min. Co-cultures were first blocked for 1 h in PBS containing 1% BSA and then were incubated overnight at 4 °C with primary antibodies in PBS containing 1% BSA and 0.2% Triton X-100. The following antibodies were used: microtubule-associated protein 2 (Map2) (Abcam, AB5392, Cambridge, UK), GFAP (Dako, Z0334), drebrin (Vinci-Biochem, BSR-M05530, Vinci, Italy), PSD95 (Cayman, 10011435), and synaptophysin (Sigma, 041M4782). Cells were finally incubated with secondary antibodies (AlexFluor Antibody, Thermo Fisher Scientific, Waltham, MA, USA) for 1 h at room-temperature. Then, 2 mg/mL Hoechst (Thermo Fisher Scientific, Waltham, MA, USA, 33342) was used to stain nuclei. ProLong Glass Antifade Mountant (Thermo Fisher Scientific) was used as a mounting agent.