Polyethylene glycol

All sacrifices were done in the afternoon, approximately 24 h after each mouse’s final instillation. Larger blocks of mice were sacrificed over two days so that sacrifices could be done in a narrow time window to control for possible circadian effects. After about 6 weeks of instillations, mice were deeply anesthetized with ketamine:xylazine (200 μg:10 μg/g body weight ip) and then intracardially perfused with 30 ml of 0.1 M phosphate buffered saline (PBS) followed by 30 ml of 4% paraformaldehyde in PBS and post-fixed in 4% paraformaldehyde in PBS for 1 h. Brains were transferred to PBS overnight, dehydrated in increasing concentrations of ethanol and embedded in polyethylene glycol (Polysciences), sectioned sagittally at 6 μm on a rotary microtome, mounted onto Superfrost++ slides, dried overnight and stored at −20 °C. Series of every fifth section throughout the dorsomedial hippocampus were processed for quantification of cells containing Iba-1 (expressed in microglia), the inflammatory cytokine IL-1β, doublecortin (DCX, expressed in immature neurons), BrdU and the neuronal marker Hu (expressed in neurons of all ages) or NeuN (expressed in mature neurons).
All IHC protocols were run with a negative control slide omitting primary antibody, and equivalent numbers of sections for each treatment were run in each assay. All rinses were 10 min unless otherwise stated.