Dmi4000 fluorescence microscope

Measurements were performed as we previously described (Velazco-Cruz et al., 2019 (link)). Stage 6 clusters were single-cell dispersed with trypLE, plated overnight, and fixed with 4% paraformaldehyde (Electron Microscopy Science; 15714) for 30 min at RT. Samples were treated for 30 min with blocking/permeabilizing/staining solution (5% donkey serum (Jackson Immunoresearch; 017-000-121) and 0.1% Triton X-100 (Acros Organics; 327371000) in PBS). Samples were then incubated overnight at 4°C with primary antibody diluted in staining solution, incubated 2 hr at 4°C with secondary antibodies diluted in staining solution, and stained with DAPI for 5 min. Nikon A1Rsi confocal microscope or Leica DMI4000 fluorescence microscope were used to take images. The antibodies used are listed in Table S4.

SC-β cells and human islets were retrieved from the engrafted
devices as mentioned above. The SC-β cells or human islets were extracted
as mentioned before, shipped overnight, and cultured in S6 or human islet media
(CMRLS with 10% FBS) for recovery of SC-islets or human islets, respectively.
The clusters were single-cell dispersed with TrypLE for 10 min and seeded in
Matrigel-coated 96 well plates (Cellvis, 963-1.5H-N) overnight for attachment in
their respective recovery media. Cells were washed with 2 mM glucose KRB and
incubated with 20 μM Fluo-4 AM (Invitrogen; F14201) in 2mM glucose for 45
min at 37°C in incubator. Next, the cells were washed with 2 mM glucose
KRB and challenged with 2 mM glucose KRB, 20 mM glucose KRB, and 20mM glucose 30
mM KCl KRB, sequentially. Images were taken every minute using a Leica DMI4000
fluorescence microscope and calcium flux was calculated with ImageJ.

Immunohistochemistry was performed as described previously^{47 (link)}. Three rats from each
treated group as indicated above (four groups; N = 3 rats/group) were
anesthetized with isoflurane and perfused with 300 ml of 4% paraformaldehyde
in 0.1 M phosphate-buffered saline (PBS; pH 7.4). After perfusion, sciatic
nerves or adjacent muscles were dissected, postfixed at 4°C for 4
hours and cryoprotected in 30% sucrose overnight. The transverse or
longitudinal sections were cut on a cryostat at a thickness of 20 μm
and collected from each tissue by grouping every third sections. The
sections were first blocked for 1 hour at 25°C in 0.01M PBS
containing 5% goat serum and 0.3% Triton X-100 and then incubated overnight
at 4°C with mouse anti-myelin basic protein (MBP; RRID: AB_10120129;
catalog number: SMI-99P; lot number: 808401. 1:500; BioLegend, San Diego,
CA, USA)^{17 (link)} or rabbit
anti-CD68 (RRID: AB_10975465; catalog number: ab125212; lot number: 43780.
1:800; Abcam, Cambridge, MA, USA)^{56 (link)}. The sections were finally incubated with goat
anti–mouse or anti-rabbit antibody conjugated to Cy2 or Cy3 (1:500;
Jackson ImmunoResearch, West Grove, PA, USA) for 2 hours at room
temperature. Immunofluorescence-labeled images were randomly taken using a
Leica DMI4000 fluorescence microscope with a DFC365FX camera (Leica,
Germany). At least 5 sections per rat (15 sections/group) were examined.