Millicell membrane inserts

Acute cerebral cortex slices were obtained from a mixed population of 4-day-old female and male Wistar rats. The animals were anaesthetised by isoflurane inhalation and decapitated. Brains were isolated, the brain hemispheres were dissected in DMEM medium (Merck) and sliced at a thickness of 400 μm perpendicularly to their longitudinal axis using the McIlwain tissue chopper (Plano, Wetzlar, Germany). Slices were placed onto Millicell membrane inserts (Merck) and transferred into six-well plates with 1 mL of nutrition medium per well (25% heat-inactivated horse serum, 25% HBSS, 50% DMEM, 2 mM glutamine, pH 7.2). Slices were maintained under standard conditions of 37 °C, 100% humidity and 5% CO₂ for a period of 24 h prior to hypoxia.

CX3CR1-GFP⁺ microglia were harvested from E15 CX3CR1-GFP mice using a cell sorter (FACS SORP Aria II) and then suspended in saline at a density of 1.0 × 10⁴ cells µl⁻¹. The cells were transplanted into the CP by injection through a glass capillary into isolated E15 ICR brain from the backside of the forebrain, just beneath the meninges; the capillary was pulled backward while the cells were ejected. For the control, only saline was injected into the brains. After transplantation, brains were embedded in 2% agarose gel and then sliced coronally (400 μm thick). The slices were cultured on Millicell membrane inserts (0.4 μm) (Merck Millipore) placed on a 35 mm culture dish for 24 h at 37 °C, and then fixed and subjected to immunohistochemistry (Supplementary Fig. 12).

Slice cultures of the hippocampus were made as previously described (Stoppini et al., 1991 (link); Nishimura et al., 2008 (link)). Briefly, the day of birth was designated as postnatal day 0 and transverse hippocampal slice cultures were prepared from 5-d-old control CaNB1wild-type mice (WT/WT, Thy1GFP-M/Thy1GFP-M) and from littermate fCaNB1 mice (fCaNB1/fCaNB1, Thy1GFP-M/Thy1GFP-M). Animals were deeply anesthetized with isoflurane vapors, and the forebrain was removed and submerged in cooled Leibovitz’s L-15 culture media (Invitrogen). The hippocampi were dissected free of adhering tissue and placed on a Teflon stage of a custom-built mechanical tissue chopper, with a small amount of L-15 media. Transverse (375 µm) slices were cut and placed on warmed Millicell membrane inserts (Millipore) presoaked in normal culture media consisting of 98% Neurobasal A (Invitrogen), 2% B27 (Invitrogen), and 0.5 mM glutamine (Sigma). Three slices were put on each insert and arranged such that they did not contact one another. The Millicell membranes were then placed in six-well plates and placed in a water-jacketed incubator at 37°C with 5% CO₂ atmosphere. The culture media were changed at 2-d intervals. Following 3 or 4 d in vitro (DIV), randomly selected slices were treated with 100 µM bicuculline methobromide (Sigma).