10x plan apochromat 10x 0.45na air objective

PFA-fixed neuronal cultures and mouse brain slices were imaged using a Zeiss 710 laser scanning confocal microscope running Zen Black software (Zeiss Microscopy). Fluorescence was acquired using 405/449 nm (DAPI), 488/515 nm (Alexa 488), and 633/670 (Alexa 647) nm laser excitation/emission wavelengths. Hippocampal sections were imaged on a Zeiss 10x Plan-Apochromat 10x/0.45NA air objective capturing 0.83 μm/pixel in x and y dimensions. Z-stacks were 20.83 μm tall with a 5.21 μm step size. For cultured neurons, a Zeiss 63× Plan-Apochromat/1.4NA oil objective was used to capture 2D planes yielding 0.13 μm/pixel in x and y dimensions. Z-stacks were captured at 3.36 mm tall using 0.42 μm z-steps, and max intensity projections were used for analysis. Analysis was performed in ImageJ (NIH). Spine and dendrite fluorescence intensities were measured using ROI masks of all clearly identifiable mushroom-shaped spines and dendrite segments closest to the spine of interest. Mean fluorescence intensity of spines and dendrites for each neuron were used to generate spine/dendrite ratios. Spine/dendrite ratios were consistent across the entire range of fluorescence intensities for Cav2.3-EGFP (R² = 0.086)), Kv4.2-Myc (R² = 0.076), and mCherry (R² = 0.021).