Antibodies used for imaging-mass cytometry are listed in Supplementary Table 3 .
Purified antibodies lacking carrier protein were conjugated with metal reporters
by using a MaxPar X8 Antibody Labeling Kit (Fluidigm). Snap-frozen human fetal
intestinal biopsies were sectioned at a thickness of 5 μm and fixed by
incubating with 1% paraformaldehyde for 5 min at rT followed by 100% methanol
for 5 min at –20 °C. After fixation, tissue sections were washed
in Dulbecco’s phosphate-buffered saline (Thermo Fisher Scientific)
containing 0.5% bovine serum albumin and 0.05% Tween, rehydrated in
additive-free Dulbecco’s phosphate-buffered saline, washed again, and
blocked with Superblock Solution (Thermo Fisher Scientific). Tissue sections
were then stained with a cocktail of metal-conjugated antibodies overnight at 4
°C, washed, and incubated with 125 nM Cell-ID Intercalator-Ir for 30 min
at rT. After a further wash, tissue sections were dipped in Milli-Q water (Merck
Millipore) for 1 min and dried for 20 min at rT. Data were acquired using a
Hyperion™ imaging-mass cytometer (Fluidigm) at a resolution of 1
µm, with settings aligned to company guidelines. The ablation frequency
was 200 Hz, and the energy was 6 dB. Regions of interest were acquired at a size
of 1 by 1 mm2. All data were stored as MCD files and txt files.
Purified antibodies lacking carrier protein were conjugated with metal reporters
by using a MaxPar X8 Antibody Labeling Kit (Fluidigm). Snap-frozen human fetal
intestinal biopsies were sectioned at a thickness of 5 μm and fixed by
incubating with 1% paraformaldehyde for 5 min at rT followed by 100% methanol
for 5 min at –20 °C. After fixation, tissue sections were washed
in Dulbecco’s phosphate-buffered saline (Thermo Fisher Scientific)
containing 0.5% bovine serum albumin and 0.05% Tween, rehydrated in
additive-free Dulbecco’s phosphate-buffered saline, washed again, and
blocked with Superblock Solution (Thermo Fisher Scientific). Tissue sections
were then stained with a cocktail of metal-conjugated antibodies overnight at 4
°C, washed, and incubated with 125 nM Cell-ID Intercalator-Ir for 30 min
at rT. After a further wash, tissue sections were dipped in Milli-Q water (Merck
Millipore) for 1 min and dried for 20 min at rT. Data were acquired using a
Hyperion™ imaging-mass cytometer (Fluidigm) at a resolution of 1
µm, with settings aligned to company guidelines. The ablation frequency
was 200 Hz, and the energy was 6 dB. Regions of interest were acquired at a size
of 1 by 1 mm2. All data were stored as MCD files and txt files.