Neutral buffered formalin

Mice lungs were collected at 4 or 14 days post-infection (dpi), halved and fixed in 10% neutral buffered formalin (Sigma Aldrich) for at least 16 h before they were dehydrated, embedded in paraffin and prepared into 5 micron sections. Lung sections were stained with Haematoxylin and Eosin (H&E), and examined for the presence of histopathological abnormalities.
The remaining lung halves collected 4 dpi were immersed into 1 ml of PBS, homogenized in whirl-pak sampling bags (Sigma Aldrich), transferred into microfuge tubes and centrifuged at 6000x g for 10 min at 4°C. Supernatant fractions were then transferred into fresh tubes and stored at −80°C. To perform virus plaque assays, supernatants were thawed, serially diluted with PBS and applied onto MDCK cells at 90% confluency for 1 h at 37°C to allow virus adsorption. Cells were subsequently rinsed with pre-warmed PBS and cultured in MEM (Thermo Fisher Scientific) supplemented with 1.2% Avicel® (FMC Biopolymer) and 2.5 µg/ml trypsin (Thermo Fisher Scientific). After 2 days of incubation, cells were fixed with 10% neutral buffered formalin for at least 16 h before plaques were visualized and quantified by staining with 0.1% crystal violet solution (Sigma Aldrich).

Sample A was freshly post-fixed for 24 h at RT in 10% neutral buffered formalin (Merck KGaA) after surgery, while Sample B was snap-frozen in liquid N₂ and stored at –80 °C until further use. Upon thawing, Sample B was postfixed as described above. Then both samples were rinsed in 1× PBS, dehydrated, and embedded in paraffin using standard procedures. 5 µm sections were cut using a sliding microtome. RNAscope assay was performed on 5 µm thick sections using RNAscope Multiplex Fluorescent Reagent Kit v. 2 (Advanced Cell Diagnostics) according to the manufacturer’s protocol hybridizing with human SSTR4 (red) and VGLUT1 (green) probes. RNAscope 3-plex human positive and negative control probes were used in parallel to ensure interpretable results. Probes, applied dilutions of fluorophores are listed in Table S3.

Either the entire intestinal tract was removed and divided into small
intestine (proximal, middle, and distal) and colon, or in case of
wounding/colitis experiments, only the colon was removed. The intestines were
flushed with phosphate-buffered saline, opened longitudinally using a gut
preparation apparatus, and fixed overnight in 10% neutral buffered formalin
(Merck, Readington, NJ). The tissues were then embedded in paraffin and
sectioned at 4 μm using a microtome (Leica, Buffalo
Grove, IL) with diethylpyrocarbonate-treated water (Merck) in the water
bath.
To find the ulcers generated by biopsy wounding, the entire tissue block
was sectioned, and 4 serial sections collected, followed by a
50-μm trim (discarded), throughout the block.
H&E staining was performed on 1 of 4 sections of each series following
standard procedures, leaving 3 blank slides per series for other stains. The
mid-ulcer series of sections was determined for all ulcers by identifying their
range within the entire set of H&E slides per block. For whole-mount
scanning, colons were washed with cold phosphate-buffered saline, whole-mounted,
and fixed in 4% paraformaldehyde for 3 hours at room temperature. In situ
hybridization, immunohistochemistry, multiplex, and whole-mount staining were
completed using standard techniques described in detail in the Supplementary
Methods.

Recipient rectum and colon tissue without stool were preserved in 10% neutral buffered formalin (Merck, Germany), and Haemotoxylin and Eosin (H&E) staining and digital images via a 20x slide scanning were processed by the Histology Department of WEHI. Histology scores were given to the gut tissues according to the number of apoptotic cells, mucosal integrity, and lymphocyte infiltration (each scored out of 3), by an independent, blinded pathologist.