Normal horse serum

Tissue was then snap frozen in OCT Tissue-Tek freezing medium (Sakura). Cryostat sections of 7 µm were air dried, fixed with acetone, and washed in DPBS. Blocking was performed with 20% normal horse serum (PAA Laboratories Inc.) for 15 min at room temperature. Sections were incubated with biotin-conjugated anti-mouse IgM (clone 11–41, BD Biosciences), followed by streptavidin Alexa Fluor 594 (Life Technologies) or FITC-conjugated anti-mouse monocyte and macrophage (MOMA; Abcam). Negative controls were no primary antibody or stained with isotype-matched antibody. Images were acquired on an Olympus BX51 microscope using Micro-Manager software (Vale Laboratory).

OSCs were prepared as described by [45 (link)]. In brief, brains from 2 to 3 month old C57BL/6 mice were removed and immediately immersed in ice-cold Ringer solution [2.5 mM KCl (Merck KGaA, Darmstadt, Germany), 1 mM MgCl₂ (Sigma-Aldrich), 260 mM d-Glucose (Applichem), 26 mM NaHCO₃ (Merck KGaA), 1.25 mM NaH₂PO₄ (Merck KGaA), 2 mM pyruvic acid (PAA Laboratories), 3 mM myo-inositol (Sigma-Aldrich), 1 mM kynuric acid (Sigma-Aldrich), 2 mM CaCl₂ (Carl Roth GmbH), pH 7.3]. The brains were embedded in 4% low melting agarose (Sigma-Aldrich). After polymerization agarose blocks were trimmed and glued onto the cutting table of a vibratome (VT1000, Leica, Heerbrugg, Switzerland). Brains were cut in coronal slices of 250 µm. Slices were then collected and stored in ice-cold Ringer solution before floating onto semi-porous membrane inserts (Millipore, 0.4 mm pore diameter) according to Stoppini et al. [46 (link)]. The slices were cultivated in wells of a 6-well plate at 37°C under 5% CO₂ in a standard medium consisting of DMEM/Ham’s F12 (pH 7.3; PAA Laboratories), 24% normal horse serum (PAA Laboratories), 2% HEPES (PAA Laboratories), 0.1% gentamycin (PAA Laboratories) and additional 10 mM d-glucose (Sigma-Aldrich). Medium was changed every other day and viability of brain slices was assessed using PI staining.