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Figure 1. Frequency of microglial-AIS contact is not altered in LPS-induced neuroinflammation. Female c57black6 mice were given a single intraperitoneal injection of LPS (5 mg/kg) or vehicle (0.9% saline, 10 mL/kg). Confocal z-stacks spanning an optical thickness of 25 μm, using a pinhole of 1 Airy disc unit and Nyquist sampling (optical slice thickness, 0.48 μm), were collected from neocortical layer V for each of six sections (spanning 1.1 mm anterior to the bregma to 2.5 mm posterior to the bregma) per mouse, resulting in 12 images per animal (n = 4-6 animals per treatment group). Microglial-AIS contact was quantitatively analyzed at 6 h-2 weeks post-injection in a blind manner using Volocity™ 3D Image Analysis Software, allowing each confocal z-stack to be observed in three dimensions. The number of microglia, AISs, and contact points in each double immunolabeled z-stack was counted manually. Contact points along the six edges of the z-stacks were excluded from analysis. A-F: double immunolabeling of Iba-1 and AnkG revealed that microglia (Iba-1, green) contact AISs (AnkG, red) (white arrows) in the cortex of saline- and LPS-injected mice; G: the mean ± SEM of microglia making AIS contact per FOV in saline- and LPS-treated mice as a percent of saline controls. Quantitation of confocal z-stacks revealed that ~45% of microglia contact AISs in the cortex of saline-injected control mice. Contact was defined by co-localization of Iba-1 and AnkG and included process touching (A, yellow single asterisk), process alignment (A, yellow double asterisk), and process wrapping (F, yellow triple asterisk) as defined by 3D analysis. No change in the percent of microglia making contact was observed throughout the course of LPS-induced neuroinflammation; H: the mean ± SEM of the number of Microglia/FOV. A significant increase in the number of microglia was observed at 3 days post-LPS injection. Data were statistically compared by one-way ANOVA where mean differences were significant as assessed using Tukey’s post hoc analysis. An asterisk indicates a significant difference (P < 0.05) from saline. Scale bar = 20 µm. LPS: lipopolysaccharide; FOV: field of view; AISs: axon initial segments