Immunostaining and Confocal Imaging of Brain Slices

Cells were washed 3× with HBSS and fixed with 4% PFA for 20 min at room temperature. Cells were washed with PBS and glycine before addition of blocking solution (0.02% saponin, 10% normal goat serum (NGS), 1% BSA) for 1 h. Slides were incubated with primary antibodies overnight at 4 °C, washed with PBS + 0.02% saponin, and incubated with secondary antibody for 1 h [25 (link)]. Slides were mounted with Vectashield + DAPI (Vector Laboratories).
For slice culture: slices were floated in HBSS+/+ in 6-well plates containing Netwell™ Inserts (Corning). Samples were fixed in 4% PFA and 0.1% Triton X-100 for 1 h, rinsed 3× in PBS, then treated for 10 min of 1.5 mg/ml glycine. After three washes in PBS, slices were blocked in PBS containing 5% NGS for 1 h at room temperature. Slices were labeled with primary antibody (diluted in blocking) overnight. The following day, slices were washed 3× in PBS and labeled with Alexa conjugated secondary (1:500) for 1 h. After 3 washes in PBS, slices were stained with filipin labeling solution for 2 h, washed 3× with PBS, and mounted in ProLong Gold (ThermoFisher) and imaged by confocal microscopy. Calbindin was used to outline Purkinje cells, and filipin intensity was calculated using ImageJ.