Immunocytochemistry and Quantification.

Cultured hippocampal cells were fixed for 20 min with 2% (vol/vol) paraformaldehyde, washed three times with PBS, then incubated for 30 min in a solution of 5% (vol/vol) fish skin gelatin, 0.1% Triton X-100 in PBS. Cells were incubated at room temperature for 2 h in the primary antibody solution and for 45 min with the secondary antibody solution. Each incubation was followed by three washes in PBS for 10 min each. Antibodies were diluted in 5% (vol/vol) fish skin gelatin, 0.1% Triton X-100 in PBS. Antibodies used were Thermo rabbit anti-KCNQ2 PA1-929 (1:200) and polyclonal goat anti–ank-G (1:1,000, gift from V. Bennett, Duke University, Durham, NC). Imaging was performed with a Zeiss LSM 780 confocal microscope using an oil immersion 63× objective. All images were collected at 1,024 × 1,024 pixel resolution. Quantification using NIH ImageJ software was performed based on the methods of Grubb and Burrone (38). Axon initial segments were identified as ank-G positive processes with clear axon-like morphology. Line scans down the axon defined the region of interest and the grayscale values for the desired channel (KCNQ2 or ank-G) were extracted. Sliding averages of the grayscale values within a 5-μm window were then normalized to the maximum value of the dataset and then plotted versus distance from the soma. The area under the curve was obtained by integration performed using OriginPro’s Baseline and Peaks function. Obtained values were compared between different shRNA treatments. In all image analysis, the experimenter was blinded to the identity of the confocal channels.