Quantifying Microglial Phagocytic Activity

The phagocytic activity of adult microglia in the hippocampus of acute brain slices was analyzed as described^{61 (link)}. Brains from LysM-Cre⁺/GRN^F/F or LysM-Cre⁻/GRN^F/F mice were washed in carbogen-saturated (95% O₂, 5% CO₂) artificial cerebrospinal fluid (ACSF) containing (in mM): NaCl 126; KCl 2.5; MgSO₄ 1.3; CaCl₂ 2.5; NaH₂PO₄ 1.25; NaHCO₃ 26; D-glucose 10; pH 7.4 (all from Sigma). Coronal slices (130 μm) were prepared using a vibratome (Microm, Walldorf, Germany) at 4°C, and allowed to rest in ACSF buffer at room temperature for 1 h before incubation with red fluorescent carboxylated microspheres (1 μm diameter, FluoSpheres^®, Invitrogen, 1:100) in PBS (Cellgro) containing 4.5 g l⁻¹ D-glucose (Sigma) for 60 min at 37°C. The slices were washed and fixed with 4% PFA. To visualize microglia, slices were permeabilized (2% Triton-X, 2% BSA, 10% donkey serum in PBS) and incubated with anti-Iba-1 (1:750, Wako), followed by donkey anti-rabbit Alexa 488 (1:250, Invitrogen) and Hoechst 33258 (Sigma-Aldrich; 1:10000). Imaging was performed with a Nikon ECLIPSE Ti 2000 spinning disk confocal microscope with a 20x objective. Slice-spanning Z-stack images (1 μm step-size) were acquired and analyzed using ImageJ MacBiophotonics cell counter plugin.