Lucifer yellow ch dilithium salt

Transepithelial resistance (TEER) was measured with an EVOM2 Volt/Ohm meter using STX2 electrodes (World Precision Instruments, Sarasota, United States). To assure the electrodes were fully submerged in medium, the media volumes were adapted to 100 μL apical and 700 μL basolateral before the first TEER measurements were performed. The TEER values after BA exposure were expressed as percentage of the TEER value measured just before BA exposure. After 24 h of BA exposure, culture inserts were washed twice with PBS and transferred to a new 24-wells plate. Lucifer Yellow CH dilithium salt (L0259, Sigma) was dissolved in phenol red-free medium (Gibco) to 1 mg/mL and 100 μL was added to the apical compartment. In the basolateral compartment, 700 μL phenol red-free DMEM was added and afterwards the plate was incubated at 37 ​°C/5% CO₂ for 3 h. Subsequently, 100 μL of the basolateral compartment was collected and fluorescence was measured at 425/515 nm (excitation/emission). An empty cell culture insert served as a control for complete paracellular permeability.

ZCL278 was purchased from Tocris and diluted in DMSO at 50 mM. ICAM-1 recombinant protein (#ADP4-050), and fibronectin isolated from human plasma (1918-FN-02M) were purchased from R&D Systems. The type I solution of rat collagen, Lucifer Yellow CH dilithium salt, Resveratrol, and Lithium Chloride were purchased from Sigma-Merck. The recombinant human CCL2/MCP-1 Protein was obtained from R&D Systems and Miltenyi Biotec.

For intracellular staining, brains were dissected in bee physiological saline solution (NaCl (130 mM), KCl (6 mM), MgCl₂ (4 mM), CaCl₂ (5 mM), glucose (25 mM), sucrose (170 mM) adjusted to pH 6.7 using diluted HCL) and fixed overnight at 4°C in 4% paraformaldehyde (PFA, Roth, Karlsruhe, Germany) in 0.1 M phosphate-buffered saline saline (PBS; NaCl (37 mM), KCl (2.7 mM), Na₂HPo₄ (8 mM), KH₂Po₄ (1.4 mM), adjusted to pH 6.7 using diluted HCL). Brains were washed three times in PBS for 10 min. If neurons were filled with Neurobiotin, brains were preincubated at room temperature for 2 h in 0.3% Triton X (Sigma-Aldrich, München, Germany) in PBS and incubated over night at 4°C in Streptavidin-Cy5 (Dianova, Hamburg, Germany) and sodium azide (0.005%). Lucifer Yellow (Lucifer Yellow CH dilithium salt, Sigma-Aldrich, München, Germany, 1:100 in PBS, respectively) was added for neuropil staining. On the next day, the brains were washed in PBS (for 15, 30, and 45 min) before they were dehydrated in an ascending ethanol series (each 10 min in 50, 70, 90, 99, and 100% ethanol). Subsequently the brains were cleared and mounted in methyl salicylate (Roth, Karlsruhe, Germany).