Hc pl apo cs2 40x 1.30 oil

Cells were plated at 25,000 cells/cm² overnight on round 12 mm No. 1.5 coverslips in a 24-well plate overnight, treated with or without 0.5 µM AP for 24 h, and fixed–stained as with immunofluorescence up to the primary antibody step (Supplementary Data 7). Proximity ligation was performed with the Duolink In Situ Orange Starter Kit Mouse/Rabbit (Millipore Sigma, DUO92102-1KT) according to the manufacturer’s recommendation. Samples were imaged in photon counting mode on a Leica STELLARIS 8 confocal microscope with a 40x objective (HC PL APO CS2 40x/1.30 OIL), 142 nm step size, 0.425 µs pixel dwell time per imaging track, 405 nm laser (1% power) excitation and 420–566 nm bandpass emission for DAPI, and 561 nm laser (0.3% power) excitation and 566–643 nm bandpass emission for Cy3. Eleven optical sections of 1 µm step size were collected from each image field and maximum-intensity projected for analysis.

Kidney mesenchymal stromal cells (kMSCs, 2 × 10⁴ cells) and hMVECs (1 × 10⁴ cells) were mixed and seeded in 1% gelatin coated 96 well plate, cultured in EC-SFM medium (Gibco, Zwijndrecht, the Netherlands) with 1% platelet poor plasma derived serum, 30 ng/mL vascular endothelial growth factor 165 (VEGF-165) (R&D, Minneapolis, MN, USA), and 20 ng/ mL fibroblast growth factor-basic (bFGF, Miltenyi, Bergisch Gladbach, Germany). Cells were pelleted by centrifugation for 30 s at 300× g. The cells were cultured for 7 days until vascular tubes formed. Next, cells were fixed with 4% PFA and 0.2% Triton-X100 in PBS for 10 min at room temperature, washed with PBS, and blocked for 1 h at room temperature in 5% BSA in PBS. Cells were incubated overnight at 4 °C with primary mouse anti-human CD31 (555445, BD Biosciences, San Diego, CA, USA) and alpha-SMA (C6198, Sigma, Zwijndrecht, the Netherlands), followed by an appropriate secondary antibodies and Hoechst 33528 for 1 h, all in blocking buffer. Cells were examined using a LEICA TCS SP8 X WLL (Leica, Rijswijk, The Netherlands) and a 60× objective (HC PL APO CS2 40x/1.30 OIL, Leica). Sequential 16-bit confocal images (xyz dimensions, 0.142 × 0.142 × 0.3 μm) were recorded using LAS-X Image software (Leica) and analyzed with ImageJ. Both vascular area and vascular branches were quantified.

Brains of 17-day-old animals raised at 29 °C were dissected and fixed in 4% paraformaldehyde in PBS for 1.5 h at RT. Images were acquired using a Leica TCS SP8 confocal laser scanning microscope, using the objective HC PL APO CS2 40x/1.30 OIL, numerical aperture 1.3, pinhole 999.61mAU, 600 Hz, 2048×2048 resolution and z slice distance of 0.3 µm. 30 µm stacks were scanned. Images were 3D reconstructed using Bitplane Imaris. An area of 512×512 pixels was selected, reconstruction was carried out for 13,5 µm stacks starting at 7,5 µm and ending at 21 µm of respective 30 µm stacks. Parameters used: smoothening active and set to 0.189, local background subtraction active and set to 0.5 µm, threshold set to 4. The volume data (total volume and volume units) was extracted and quantified. Wilcoxon rank-sum test was used for statistical analysis. N indicates the number of independent experiments, while n indicates the number of individual animals. Box plots: the boundary of the box indicate the 25^th and 75^th percentile, the line within the box marks the median. Whiskers (error bars) above and below the box indicate the 90^th and 10^th percentiles. Dots indicate outliers.