Dual-channel RESOLFT nanoscopy by fluorescence kinetics

Hippocampal brain slices were prepared by dissecting hippocampi from postnatal day P5–P7 wildtype C57BL/6 mice, which were then sectioned in 400 μm thick slices and embedded in a plasma clot on 0.14 mm thick glass coverslips. The slices were maintained in a roller incubator at 35°C in medium containing (in ml): BME 97, HBSS 50, horse serum 50, glucose (5M) 2, glutamine (200mM) 1 — according to the method of Gähwiler et al. 1997 . Slice cultures were left to mature for 12 days in the incubator and were used in the experiments up to an age of 45 days in vitro after preparation. For transfection we injected the viruses into the CA1and CA3-regions of the slice cultures using a patch pipette connected to a pressure generator (Tooheyspritzer, Toohey Company, Fairfield, NJ, USA). The cultures were then incubated for at least 12 hours and imaged within 12–48 hours after transfection. For imaging, brain slices were transferred to an imaging chamber and maintained in artificial cerebrospinal fluid (ACSF) containing (in mM) NaCl 126, KCl 2.5, CaCl2 2.5, MgCl2 1.3, glucose 30 and HEPES 27; the pH was adjusted with NaOH to 7.4.