Stellaris 8 confocal microscope

EndoC-βH1 cells were plated in Nunc Lab-Tek II Chamber Slides (Thermo Fisher Scientific) and fixed with 3% PFA-K-PIPES and 3% PFA-Na₂BO₄ for 5 and 10 min, respectively, followed by permeabilization with 0.1% Triton X-100 for 30 min at room temperature (all Sigma-Aldrich). The cells were incubated in blocking solution containing 5% donkey serum (Jackson ImmunoResearch) in PBS for 30 min. Primary antibodies were diluted in blocking solution and slides incubated overnight at 4 °C followed by incubation with secondary Alexa Fluor-conjugated antibodies and DAPI (MP Biomedicals) at room temperature for 1 h (Supplementary Table 4). Imaging was performed on a STELLARIS 8 Confocal Microscope (Leica Microsystems).

Light microscopy images were taken with a Leica 2500 microscope (20× and 40× objectives). Fluorescent markers were imaged with a Leica Stellaris 8 confocal microscope (63× objective). Confocal images were obtained with Leica Las AF software using PBS or water as the imaging medium. All confocal images with multiple channels were imaged in sequential scan mode. Confocal settings may have varied between experiments but always stayed the same for the experimental sample and the respective control. To better optimize the SR2200 cell wall staining, the signal was sometimes adjusted during imaging and may thus vary between the sample and control.
The Leica Stellaris 8 has a Tau-gating mode that makes it possible to separate GFP signals from background signals. GFP markers were always imaged with this Tau-gating mode, gathering signals between 1.3 ns and 9 ns.
Contrast was adjusted with Abobe Photoshop for the whole GUS images in the following figures: Figs. 1f–h and 5g. A transparent box was added to the figure to make the text more readable in the following figures: Figs. 1a,f–h,j, 2a–d, 3a,b,f,h, 4a–c,g and 5a,b,d–g,j–m and Extended Data Fig. 3a–d,f,g.

For the confocal microscopy studies, POPC:POPG (1:1) GUVs were prepared with 2% NBD-DPPE or 0.1% Rho-DPPE GUVs, as described in Section 2.3.2. To evaluate the effect of W-BP100 on the membrane integrity and aggregation, 2% NBD-labelled GUVs were used, while for the vesicle fusion assays, 2% NBD-labelled GUVs were mixed with 0.1% Rho-labelled GUVs at a 1:1 molar ratio.
GUVs were observed under confocal laser scanning microscopy in a µ-Dish 35 mm Petri dish from Ibidi® and the images were recorded on a Leica Stellaris 8 confocal microscope (Leica Microsystems, Wetzlar, Germany) equipped with the Leica Application Suite X package (LAS X, Wetzlar, Germany). GUVs (~238 μmol dm⁻³) prepared in 280 mmol dm⁻³ sucrose were first diluted five times in a 280 mmol dm⁻³ glucose aqueous solution to allow for vesicle deposition on the bottom of the Petri dishes due to a density gradient. A total of 40 µL of GUVs (~48 µmol dm⁻³) was placed on Petri dishes and small aliquots of a peptide stock solution were successively added at a concentration range of 40–120 µmol dm⁻³ (corresponding to P/L of 0, 0.8, 1.2, 2.1, 2.5). Images were acquired under a 63×-oil objective, over 10 min, with 10 s intervals between frames. Image analysis and the size of the GUV at different P/L ratios were performed using ImageJ software (version 1.53f51, U. S. National Institutes of Health, Bethesda, MD, USA).

Skin samples resulting from calcein permeation assays were fixed with formaldehyde and processed. Histological sections were obtained from skin resultant from two independent permeation assays. Briefly, tissue was paraffin-embedded and sectioned at a thickness of 5 μm. Then, hematoxylin and eosin (H&E) stained samples were analysed by optical microscopy (Inverted Optical Microscope, CK2 model, Olympus, Hamburg, Germany), equipped with a digital camera (MotiCam S12) and using 4× or 10× objectives. To further investigate pore-forming structures in the skin, unstained samples were analysed using confocal fluorescence microscopy (Leica Stellaris 8 confocal microscope, Leica Microsystems, Wetzlar, Germany) equipped with the Leica Application Suite X package (LAS X). Images were acquired, following excitation at 495 nm by a HyD X detector (510–600 nm), with a resolution of 1024 × 1024 using a 10X/0.4 objective, and then processed under ImageJ software (Fiji app, ImageJ2 version, freely available at http://imagej.net/, accessed on 27 January 2022).