Sucrose

Glutamic acid, succinic acid, adenosine-5′-diphosphate sodium salt (ADP), cytochrome c from bovine heart, malic acid, ethylene glycol-bis-(b-aminoethylether)-N,N,N′N′-tetra acetic acid (EGTA), KH₂PO₄, TrisHCl, ethylenediamine tetra-acetic acid (EDTA), amytal, carboxyatractyloside (CAT), guanosine triphosphate (GTP)quercetin, acetonitrile, 2,2-azinobis (ethyl-2,3-dihydrobenzothiazoline-6-sulphonic acid) diammonium salt (ABTS), potassium persulfate, iron(III) chloride hexahydrate (FeCl₃·6H₂O), 2,4,6-tripyridyl-s-triazine (TPTZ), hydrochloric acid, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) were obtained from “Sigma–Aldrich GmbH”, Steinheim, Germany. Sucrose, mannitol, HEPES, KCl, MgCl₂, trifluoroacetic acid, hyperoside, isoquercitrin, rutin were obtained from “Carl Roth Gmbh”, Karlsruhe, Germany.

Paraformaldehyde (VWR, International, Radnor, PA, USA) -fixed tissue was transferred to 30% sucrose (Carl Roth, Karlsruhe, Germany) solution prior to cryosectioning. Sections (5 µm) were cut, rehydrated in PBS, and blocked with 0.05% PBST (0.05% Tween-20 (VWR International, Radnor, PA, USA) in PBS) containing 10% goat serum (Szabo-Scandic, Vienna, Austria) for 30 min. Sections were incubated with anti-LAMP1 antibody (1D4B, 1:25, Developmental Studies Hybridoma Bank, Iowa City, IA, USA) overnight at 4 °C, washed with PBS, and incubated with anti-rat Alexa Fluor^® 488 secondary antibody (1:250, ThermoFisher Scientific, Waltham, MA, USA) for 2 h at room temperature. For co-staining with ORO (Sigma-Aldrich, St. Louis, MO, USA), slides were incubated for 1 h in the freshly prepared ORO staining solution and subsequently stained for 10 min with DAPI (Sigma-Aldrich, St. Louis, MO, USA) to visualize nuclei. Slides were mounted using Dako Fluorescence Mounting Medium (Dako North America Inc., Carpinteria, CA, USA) and visualized on an Olympus BX63 microscope equipped with an Olympus DP73 camera (Olympus, Shinjuku, Japan).

Tissues were sonicated twice for 10 s on ice in lysis buffer (100 mM potassium phosphate (Carl Roth, Karlsruhe, Germany), 250 mM sucrose (Carl Roth, Karlsruhe, Germany), 1 mM EDTA (ThermoFisher Scientific, Waltham, MA, USA), pH 7) and the protein amount was determined after centrifugation as described above. Lipids were extracted from 1 mg protein following Folch’s method [48 (link)]. TG, TC, and FC concentrations were determined using enzymatic kits following the manufacturer’s guidelines (DiaSys, Holzheim, Germany). CE concentrations were calculated by subtraction of FC from TC values. All lipid concentrations were normalized to protein content.

ATP, ADP, AMP, adenosine, dipyridamole, apyrase and dimethylsulfoxid (DMSO) were purchased from Sigma (Taufkirchen, Germany); 2-methylthioadenosine diphosphate trisodium salt (2meSADP) was from Tocris (Bristol, UK); DMEM, Dulbecco’s Phosphate-Buffered saline without Ca²⁺ and Mg²⁺, trypsin/EDTA solution, HEPES was obtained from Thermo Scientific (Walldorf, Germany); NaCl, KCl, MgCl₂, CaCl₂, K₂HPO₄, NaHCO₃, D-glucose, paraformaldehyde (PFA), Triton X-100, Tween 20, bovine serum albumin (BSA), and sucrose were from Roth (Karlsruhe, Germany). Heparin (Heparin-Natrium-250 000-ratiopharm) was from Ratiopharm (Ulm, Germany), Narcorene^® from Merial GmbH (Hallbergmoos, Germany), ticagrelor was purchased from Cayman Chemical (Hamburg, Germany); concentrated stock solutions of ticagrelor and dipyridamole were prepared in DMSO and diluted in artificial cerebrospinal fluid (ACSF) or cell culture medium right before use.

Whole brains were dissected from embryos from timed-pregnant C57BL/6J mice at E14.5, E16.5, E19.5 (histology only), or newborn C57BL/6J mice (P0). All animal protocols were reviewed and approved by Animal Welfare Committee at the Technische Universität Dresden and Landesdirektion Sachsen, Dresden, Germany (governmental authorities). The tissue was fixed for 24 h in 4% paraformaldehyde (Merck, Darmstadt, Germany) and kept in 30% sucrose (Carl Roth, Karlsruhe, Germany) in PBS (Thermo Fisher Scientific, Waltham, MA, USA). Then brains were snap-frozen, coronal sections at 20 μm thickness were prepared using a cryotome (Leica Biosystems, Nussloch, Germany) and mounted on Superfrost Plus slides (Thermo Fisher Scientific, Waltham, MA, USA). The slides were stored at 4°C until staining. A total of three animals per group were examined for each experiment; all data were gathered from randomly chosen fetuses from three independent litters.

As generally described in Zaqout et al. (2019) (link), decapitation was followed by rapid removal of the brain, which then was transferred into carbogenated (95% O₂; 5% CO₂) 2°C sucrose artificial cerebrospinal fluid (sACSF) containing (in mM): 85 NaCl, 2.5 KCl, 1 NaH₂PO₄, 7 MgCl₂, 26 NaHCO₃, 50 sucrose, 10 D(+)-glucose (Carl Roth, Karlsruhe, Germany), and 0.5 CaCl₂ (Merck, Darmstadt, Germany). 300 μM thick coronary slice containing somatosensory cortex were cut using a VT1200S vibratome (Leica, Wetzlar, Germany). Slices were recovered for 30 min in 34°C sACSF and were subsequently held at room temperature in modified ACSF containing (in mM): 92 NaCl, 2.5 KCl, 1.2 NaH₂PO₄, 30 NaHCO₃, 25 D(+)-glucose (Carl Roth), 5 sodium ascorbate, 20 2-[4-(2-hydroxyethyl)piperazin-1-yl] ethanesulfonic acid (HEPES), 3 sodium pyruvate, 2 MgSO₄, 2 CaCl₂ (Merck), and 2 thiourea (VWR Chemicals, Radnor, PA, United States) until recording. All variations of ACSF were continuously carbogenated.

Freeman et al. described a qualitative assay to detect the microorganisms which are able to produce biofilm [38 (link)]. This method is based on color changing of colonies on the Congo red agar (CRA) medium. Colonies with black color represent a biofilm producer whereas red-pink colonies retain non-biofilm producers. The CRA medium comprised brain heart infusion (BHI) (37 g/L) (Carl Roth GmbH + Co. KG, Karlsruhe, Germany), sucrose (50 g/L) (Carl Roth GmbH + Co. KG, Karlsruhe, Germany), agar (10 g/L) (Carl Roth GmbH + Co. KG, Karlsruhe, Germany), and Congo red dye (0.8 g/L) (Sigma-Aldrich, St. Louis, MO, USA). Congo red dye was prepared separately and added into the autoclaved BHI medium. The plates were incubated at 37 °C for 24 h aerobically.