Leica sp5 lsm

Analysis of neuron anatomy was done as previously described [99 (link)]. Briefly, after filling MSNs with a biocytin-containing intercellular solution during electrophysiological analyses, brain slices were removed from the recording chamber, fixed with 4% Histofix (Roth, Germany) and subsequently stained with Streptavidin-Alexa Fluor 594 (dilution of 1:1000; ThermoFisher Scientific Inc., Waltham, MA, USA). Slices were mounted onto slides with ProLong Gold antifade reagent (ThermoFisher Scientific Inc., Waltham, MA, USA).
Proximal and distal dendrites of MSNs in the striatum were imaged with a 63× objective (Olympus, Shinjuku, Japan) on a confocal microscope (Leica SP5 LSM; Leica, Wetzlar, Germany). Subsequent blind deconvolution was performed with Amira software (ThermoFisher Scientific Inc., Waltham, MA, USA). Whole-cell images for Sholl analysis were taken with a 40× objective (Olympus, Shinjuku, Japan).
Semi-automatic spine counting of proximal and distal dendrites and tracing of dendrites of whole cells was carried out with NeuronStudio (version 0.9.92; Computational Neurobiology and Imaging Center Mount Sinai School of Medicine, New York, NY, USA). Sholl analyses of the reconstructed neurons were performed with Neurolucida (version 3.70.2; MBF Bioscience; Williston, VT, USA), with starting radius and radius increment parameter set to 10 µm.

Mice were deeply anesthetized with isoflurane and transcardially perfused with phosphate buffered saline (PBS) and 4% PFA (Roth, Karlsruhe, Germany). Then, the brain was extracted and cut into coronal mouse brain slices (70 µm thick) with a tissue slicer (VT1000; Leica, Wetzlar, Germany) containing the striatum were fixed in 4% Histofix (Roth, Germany) and stained with an antibody detecting exclusively huntingtin aggregates as inclusions (monoclonal MW8, dilution 1:10 in 3% BSA, 2% NGS, 0.2% Triton-X-100, PBS buffer corresponding to 5 µg/mL; Developmental Studies Hybridoma Bank, Iowa City, IA, USA). Subsequently, after several washing steps with PBS, the secondary antibody Rabbit anti-Mouse IgG (H + L) Alexa Fluor 594 was added (A27027, dilution of 1:1000, ThermoFisher Scientific Inc., Waltham, MA, USA). Nuclei were visualized using a DAPI staining (D1306, dilution of 1:10,000; ThermoFisher Scientific Inc., Waltham, MA, USA). After several washing steps with PBS, slices were mounted on a glass slide with ProLong Gold antifade reagent (life technologies).
Slices were imaged with a 40× objective (for whole-cell images) or a 63× objective (for dendrite close ups) objective (Leica, Wetzlar, Germany) on a confocal microscope (Leica SP5 LSM; Leica, Wetzlar, Germany).