Alexa fluorar 568 hydrazide

Individual Purkinje cells were filled with 0.02 mM Alexa FluorAR 568 hydrazide (Invitrogen, A-10441) and imaged as described previously (29 (link)). The surface area was quantified using in-house MatLab scripts and distance to first branch point measured on z-projections in Image J.

Whole-cell recordings from Purkinje cells were performed with an internal solution containing (in mM): 0.2 Lucifer Yellow (Sigma, L0144), 0.02 Alexa FluorAR 568 hydrazide (Invitrogen, A-10441), 125 K-gluconate, 15 KCl, 10 HEPES, 5 EGTA, 2 MgCl₂, 0.4 NaGTP, 2 NaATP, and 10 Na-phosphocreatine at pH 7.4. Purkinje cells were voltage-clamped at −60 mV for 25–30 min and complete cell filling was monitored by Lucifer Yellow fluorescence. Slices were then fixed with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) overnight at 4°C. Slices were washed twice in 0.1 M phosphate buffer (pH 7.4) and twice in dH₂O then stored in Vectashield (Vector Laboratories) at 4°C. Slices were wet-mounted with Vectashield onto 0.13 mm thick borosilicate glass and imaged using a Zeiss inverted LSM510 confocal microscope. Dendritic length and surface area was analyzed using ImageJ software (NIH).