Dendritic Spine Visualization in Klhl17 Mutant Mice

To visualize dendritic spines in mouse brains, Thy1-YFP mice were crossed with Klhl17 mutant mice. YFP signals were used to outline neuronal morphology. After perfusion with 4% paraformaldehyde in PBS, the brains were dissected and postfixed with 4% paraformaldehyde overnight at 4°C with gentle shaking. Fifty-μm-thick brain sections were cut using a vibratome (VT1200S, Leica). Brain slices were permeabilized with 0.3% TritonX-100 in TBS for 15 min and blocked with TNB buffer (0.5% blocking reagent in TBS, #FP1012, PerkinElmer) for 2 h at room temperature. The primary anti-synaptopodin antibody (0.2 μg/ml) was then added for 2 days at 4°C. After washing, brain slices were incubated with Alexa fluor 555-conjugated secondary antibodies for 2 h at room temperature. Details of the protocol for CA1 analysis have been described previously [53 (link)].