Ti2 spinning disk confocal microscope

Free H₂S was measured in cells using the SF₇-AM fluorescent probe [30 (link)] (Sigma-Aldrich). The probe was dissolved in anhydrous DMF at 5 mM and used at 5 μM in serum-free RPMI. Live-cell image acquisition was performed using a Nikon Ti2 spinning disk confocal microscope. Global protein persulfidation was assessed on VSMC grown on glass coverslips as previously described [9 (link)]. Cells were incubated for 20 min with 1 mM 4-Chloro-7-nitrobenzofurazan (NBF-Cl, Sigma-Aldrich) diluted in PBS. Then, cells were washed with PBS and fixed for 10 min in ice-cold methanol. Coverslips were rehydrated in PBS and incubated with 1mM NBF-Cl for 1 h at 37 °C. In parallel, a Daz2-Cy5.5 solution was prepared by mixing 1mM Daz-2, 1 mM alkyne Cy5.5, 2 mM copper(II)-TBTA, 4mM ascorbic acid and incubating overnight at RT, followed by quenching for 1h with 20mM EDTA. Fixed cells were further incubated at 37 °C for 1h in the Daz2-Cy5.5 solution. Finally, coverslips were washed 3 times in methanol and 2 times in PBS, mounted in Vectashield mounting medium with DAPI, and visualized with a 90i Nikon fluorescence microscope. Persulfidation was measured as the ratio of Daz2-Cy5.5 over NBF-Cl signal per cell by two independent experimenter blinded to the conditions using the Fiji (ImageJ 1.53t) software.

Drosophila MTs connected to guts were dissected from adult females and fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) at room temperature for 1 hour, incubated for 1 h in Blocking Buffer (5% normal donkey serum, 0.3% Triton X-100, 0.1% bovine serum albumin (BSA) in PBS), and stained with primary antibodies overnight at 4 °C in PBST (0.3% Triton X-100, 0.1% BSA in PBS). Primary antibodies were mouse anti-GFP (Invitrogen, A11120; 1:300) and mouse anti-discs-large (DSHB, 4F3,1:50). After primary antibody incubation, the tissues were washed 4 times with PBST, stained with 4′,6-diamidino-2-phenylindole (DAPI) (1:2000 dilution) and Alexa Fluor-conjugated donkey-anti-mouse (Molecular Probes, 1:1000), in PBST at room temperature for 2 h, washed 4 times with PBST, and mounted in Vectashield medium. The kidney stones can be observed under the far-red channel.
All images presented in this study are confocal images captured with a Nikon Ti2 Spinning Disk confocal microscope. Z-stacks of 15-20 images covering one layer of the epithelium from the apical to the basal side were obtained, adjusted, and assembled using NIH Fiji (ImageJ), and shown as a maximum projection.

Myotubes differentiated between 7 and 9 days were placed in an incubation chamber and maintained at 37 °C and 5% CO₂ throughout the experiment. Cells were observed using the Nikon Ti2 spinning disk confocal microscope (equipped with a SR module) through a x100 immersion objective and frames were acquired every 10 s during periods ranging from 15 min to 3 hr. The generated images were then analyzed using FIJI and movies played back at 5 frames per second (Figure 4—videos 1–4).