Professional version 19

Images were acquired on a Leica TCS SP8 DMi8 STED Inverted Confocal microscope (Leica Wetzler, Germany) using Leica Application Suite X (LASX). Z-series of 5 μm were collected beginning at the top of the tissue section inward using the following parameters: 400 Hz, z step size:0.1 um, 2048 × 2048. After acquisition, image stacks were deconvolved with Huygens Professional version 19.04 (Scientific Volume Imaging, The Netherlands, http://svi.nl) using CMLE algorithm, with SNR:20 and 40 iterations. Z stacks were exported in TIFF format for post-processing.

To visualize dendritic spines, 300-μm-thick biocytin-labeled slices were removed from the slides and stored in 0.1 mm PB. After three washes with 0.1 mm PB, the slices were embedded in 2% agarose gels and further cut into 50 μm sections with a vibratome (Leica VT1200s). Then, the 50 μm slices were remounted on slides with Fluoroshield and visualized with a Nikon C2 confocal microscope and 60× oil immersion objective (NA 1.4) at a pixel size of 0.07. Z stacks were acquired at a step interval of 0.125 μm. Images of the molecular layer of the inner dentate gyrus were taken. The original images were further deconvoluted by using Huygens Professional version 19.04 (Scientific Volume Imaging; http://svi.nl) before quantification. Then, the deconvoluted images were imported into Neurolucida software (MicroBrightField, RRID:SCR_001775) to trace the spines and quantify the total number of spines per 30 µm.