STAR 635P

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).

Slides with sorted chromosomes were incubated in blocking buffer (3% BSA, 0.5 M EDTA, Tween 20) for 1 h at RT followed by incubation with Topo IIrb12 primary antibodies (diluted 1:125) overnight at 4 °C. Afterward, slides were washed in 1 × PBS at RT and incubated with secondary anti-rabbit antibodies STAR 635P (ST635-1002-500UG, Abberior, Göttingen, Germany), diluted 1:125 in blocking buffer, for 1 h at RT. Next, slides were washed in 1 × PBS at RT, dehydrated in an ethanol gradient (70%, 90%, 100%), and mounted in two different embedding media. Samples embedded in the ProLong diamond medium (ThermoFisher Scientific/Invitrogen, Waltham, MA, USA) were cured for 24 h at room temperature in the dark before microscopy. Slides with DABCO mounting medium (Sigma-Aldrich, Darmstadt, Germany) were sealed with Fixogum rubber cement (Marabu GmbH and Co. KG, Tamm, Germany) to prevent evaporation and stored at 4 °C before microscopy.

STED images were obtained using the Abberior Quad Scan Super-Resolution Microscope. Before imaging, a laser power meter (Thorlabs) was used to measure and set the energy levels of the lasers used (approximately 1 μW for excitation, approximately 5 mW for depletion). Images (pixel size: 50 nm; 1,400 × 1,400 pixels) of endogenous SNAP25 were obtained using the Abberior STAR580 excitation and emission settings (561 nm excitation/775 nm depletion) of the Imspector software. For images of SNAP25(1100), we used the Abberior STAR635P settings (640 nm/775 nm depletion). Dwell time was set to 10.00 μs and line averaging to 2.

A simplified version of a previously published protocol was used [32 (link)]. Formaldehyde-fixed kidneys were embedded in 3% agarose in DI water and sectioned to 200 µm thickness using a vibratome. Slices were then incubated in clearing solution (200 mM boric acid, 4% SDS, pH 8.5) at 50 °C overnight. Sections were washed in PBST (0.1% Triton-X in 1X PBS, pH 7.4) for 10 min before incubation in a sheep polyclonal antibody against nephrin (R&D systems, Minneapolis, MI, USA, AF4269) diluted 1:50 in 10 mM HEPES, pH 7.5 with 200 mM NaCl and 10% TritonX-100 at 37 °C for 4 h with shaking at 500 rpm. After primary antibody incubation, samples were washed in PBST for 5 min at 37 °C and were then incubated in a donkey anti-sheep secondary antibody conjugated to Abberior STAR635P (Abberior, Goettingen, Germany, 2-0142-007-2, dilution 1:50) at 37 °C for 4 h. Samples were incubated in 80% wt/wt fructose (1 mL of dH₂O added to 4 g of fructose) at 37 °C for 15 min and then mounted in a MatTek dish with a cover slip on top, prior to imaging with a Leica SP8 3× gSTED system (Leica Biosystems, Wetzlar, Germany) using a 100× 1.4 NA objective. To quantify the slit diaphragm length per area, a previously published ImageJ macro was used [17 (link)].