C2si camera

Confocal images were obtained on a Nikon C2⁺ microscope equipped with CFI Plan Apochromat Lambda Series Objectives. Images were captured with Nikon C2si⁺ camera and analyzed using Nikon Elements software. In Thy1-GFP(M) mice, layer I of the medial PFC was identified and apical dendritic segments were imaged using 40x objective lens with a 2.5x zoom (NA 1.4) with z-stack sampling: 0.075 μm. For each sample, 6–8 dendritic segments were analyzed in NeuronStudio as previously described (Wohleb et al., 2018 (link)). Proportional area of GFAP⁺ or IBA-1⁺ material in images taken with the 20x objective was measured using standardized threshold parameters with ImageJ software (NIH, Bethesda, Maryland). IBA-1⁺ cells were counted by a trained researcher blinded to animal condition (4–6 images per sample). To quantify the number and volume of GFP + inclusions within IBA-1+ microglia (16–25 per sample) or GFAP + astrocytes, confocal images (40x; zoom 2.5x), NA 0.95, z-stack sampling: 0.2 μm) were examined in 3-dimensional space and orthogonal z-stacks with Nikon Elements Image Analysis software. Confocal imaging with these settings provides sufficient resolution to detect synaptic inclusions (average inclusion diameter: 0.25 μm) (Weinhard et al., 2018 (link)).