Rc 41

For ECS labeling, artificial cerebrospinal fluid (ACSF) was prepared from a 10× stock solution with MgCl₂ and CaCl₂ added freshly before carbogen bubbling, whereas ascorbic acid and Trolox were added after bubbling. ACSF consisted of 125 mM NaCl, 2 mM CaCl₂, 1.3 mM MgCl₂, 4.8 mM KCl, 26 mM NaHCO₃, 1.25 mM NaH₂PO₄, 7.5 mM HEPES (Gibco, 15630056), 20 mM d-glucose (Sigma, G8270-1kg), 1 mM Trolox (Sigma, 238813) and 1 mM ascorbic acid (Sigma, A5960-25G) at pH 7.4. Thereafter, fluorescent dye (Atto 643 (Atto-Tec, AD 643-25), SulfoAtto 643 or Abberior STAR 635 P (Abberior, ST635P)) was added from 5 mM stocks (dissolved in ACSF) to a final concentration of 150 µM. A 2-µl droplet of the dye-containing imaging solution was put on a no. 1.5H coverslip (Bartelt, 6.259995) that had been placed in an imaging chamber (RC-41, Warner Instruments). Using fine forceps, brain slices with the membrane attached were then carefully put onto the droplet, such that the slice was oriented toward the coverslip. A slice anchor gently kept the sample in place. Immediately afterwards, further imaging solution at room temperature was added. The imaging chamber was then placed onto the stage adaptor of the STED microscope (see below). The data in the manuscript were acquired using Atto 643 (except for Fig. 5 and Supplementary Fig. 8 (left panel) where SulfoAtto 643 was used).

Hippocampi were extracted from 5–7-day-old mice of either sex. Mouse pups were decapitated and the hippocampus isolated while the brain was submerged in ice-cold sterile filtered HBSS without Ca²⁺ and Mg²⁺ (Gibco, 14175-053) supplemented with 10 mM glucose, using a stereo microscope. The whole hippocampus was then submerged in freshly carbogenized ACSF with 150 µM Atto 643 dye and incubated for 10 min at room temperature with gentle agitation. Afterwards, entire hippocampi were placed on a no. 1.5H coverslip that had been placed in an imaging chamber (RC-41, Warner Instruments) with the alveus region facing the coverslip. A slice anchor gently kept the sample in place when freshly carbogenized ACSF with 150 µM Atto 643 dye was added for imaging. The imaging chamber was then placed onto the stage adaptor of the STED microscope.