S100+ cells: A new neuro-immune cross-talkers in lymph organs
Supplementary figure
Supplementary Fig. 1 Anterograde tracking experiment
These rats were anesthetized with pentobarbital sodium (20 mg/kg, i.p.), then 1 μl of 3%
Fluoro-Ruby in distilled water was injected into the superior cervical ganglion by using Hamilton
microsyringe with a removable injection needle (24 gauge, Hamilton, Co., USA). After survival
for 14 days, the ipsilateral cervical lymph nodes were removed. Frozen sections (15-25 µm) were
cut in cross-section on a frozen microtome and picked up on gelatin-coated glass slides and
observed under Confocal microscope.
Supplementary Fig. 2 Control experiment
In order to prove the labeling of sympathetic nerve fibers is the result of axonal transport of FR
dye in neurons, not as the diffusion FR dye, we have done this control experiment. After cutting
superior cervical ganglion, 1 μl of 3% Fluoro-Ruby was injected into the point of removing
ganglion. The animal was allowed to recover and was sacrificed typically 9-14 days later. The
animal was then perfused with neutral buffered formaldehyde (10% formalin or 4%
paraformaldehyde in 0.1 M neutral phosphate buffer.) The cervical lymph nodes were removed
and postfixed for at least overnight in the same fixative solution plus 20% sucrose for
cryo-protection. The lymph nodes were then cut on a freezing sliding microtome or cryostat into
sections usually between 15 and 20 microns in thickness. The results showed that Fluoro-Ruby
distributes in the diffusion between cells, and not surrounds the cells as experimental group.
Supplementary Fig. 3 The results of retrograde tracing
We also retrograde tracing the sympathetic neurons in SCG by injected Fluoro-Gold to cervical
lymph nodes. The labeled cells in SCG can be seen (arrows in A and B).This experiment proved
that the sympathetic nerve fibers projected from SCG directly contact with some cells in cervical
lymph node.
Supplementary Fig. 4
Ultrastructural of lymph node: the electron micrograph shown the presence of unmyelinated axons
adjacent to the innervated cells (arrows in A, B). There is a gap junction between this axon
endings and cytomembrane, partially synaptic vesicles, mitochondria and smooth endoplasmic
reticulum rich in denuded axon endings, rich in (arrows in C and D). These results give an indirect
evidence of synaptic structure in lymph organs.
Movie 1:The three-dimensional reconstructions of the labeled nerve fibers by FR and
cell membrane staining: Red fluorescence is labeled nerve fibers, and green
fluorescence is labeled the plasma-membrane with fluorescent dye DiO.
Movie 2: This is a partial enlarged film from movie 1, Red fluorescence is labeled
nerve fibers, and green fluorescence is labeled the plasma-membrane with fluorescent
dye DiO. This movie clearly showed 0.1-0.2μm labeled nerve ending in cell
membrane.