Neutral buffered formaldehyde

Cells were fixed in 4% neutral-buffered formaldehyde (Sigma-Aldrich) and permeabilized and blocked in 10% donkey serum (Sigma-Aldrich) in 0.1% Triton X-100 in DPBS (PBST) for 20 minutes at RT. Cells were incubated with primary antibodies diluted in 1% donkey serum in PBST for 1 hour at RT before they were washed 3 times in DPBS. Appropriate Alexa-fluor-conjugated secondary antibodies diluted 1:1000 in 1% donkey serum in DPBS were applied to the cells for 30 minutes in the dark at RT. Antibodies used in this study are listed in Table S3. Cells were then washed 3 more times in DPBS. DAPI was diluted 1:10,000 in DPBS, added to the cells and incubated for 2 minutes at RT in the dark. Cells were washed a further 3 times with DPBS and then imaged using the EVOS FL imaging system (Life Technologies).

Lung right caudal lobes were slowly filled and fixed with 4% neutral buffered formaldehyde (Sigma Aldrich, Saint Quentin Fallavier, France). Following dehydration, they were embedded in paraffin (Richard-Allan Scientific ^®, Thermo Fisher Scientific France, Illkirch-Graffenstaden, France). Then, tissue blocks were sectioned and slides were stained using hematoxylin and eosin staining technique prior to bright field microscopy histopathology analysis (Merck ^®, Fontenay Sous Bois, France). The histopathological assessment of the lung slides was performed by board-certified anatomic pathologists.

Pathogen-free, male, juvenile, wild-type (WT) C57BL/6J mice and female, adult, NSG™ mice (engrafted with human CD34⁺ hematopoietic stem cells) were purchased from Jackson Laboratories (Ban Harbor, ME, USA). All mice experiments were conducted strictly following the National Institutes of Health (NIH) guidelines for humane care and use of laboratory animals and local Institutional Animal Care and Use Committee (IACUC) standards. The animal experimental protocols for this study were approved by the University of South Carolina (Columbia, SC, United States) and in compliance with the ARRIVE guidelines.
Upon arrival, all mice were housed in a 22–24 °C temperature-controlled room with a 12 h light/12 h dark cycle and had ad libitum access to food and water. Upon completion of dosing, all mice were sacrificed. The distal parts of the small intestine were collected from each sacrificed mouse and immediately fixed in 10% neutral buffered formaldehyde (Sigma-Aldrich, St. Louis, MO, USA). Also, serum samples were prepared from freshly collected blood and were kept at − 80 °C. Fecal pellets were collected from the colon of each mouse, snap-frozen immediately, and preserved at − 80 °C for microbiome analysis.

Right caudal lung lobes were slowly filled and fixed with 4% neutral buffered formaldehyde (Sigma Aldrich, St. Louis, MI, USA). Following dehydration, they were embedded in paraffin. Then, tissue blocks were sectioned and slides were stained using hematoxylin and eosin or Sirius red prior to analysis under bright-field microscopy.

Adult (10 weeks old), wild-type, pathogen-free, male C57BL/6J mice were purchased from Jackson Laboratories (Bar Harbour, ME, USA). Mice experiments were conducted maintaining the National Institutes of Health (NIH) guideline for human care and use of laboratory animals and local Institutional Animal Care and Use Committee (IACUC) standards (Animal Use Protocol reference number: 101345; Protocol Number: 2419-101345-072318; approval date: 23 July 2020). The animal handling procedures were approved by the University of South Carolina (Columbia, SC, USA). Upon arrival, all mice were housed in a temperature (22–24 °C) and humidity-controlled animal room and had ad libitum access to chow diet and water with a 12-h light/12-h dark cycle. Mice were sacrificed after completion of the dosage regimen. Immediately after anesthetization, blood was collected from mice using the cardiac puncture method, and serum was extracted from it. All serum samples were preserved at −80 °C until further analysis was performed. Organs, including the distal parts of the small intestine, were collected and fixed in 10% neutral buffered formaldehyde (Sigma-Aldrich, St. Louis, MO, USA), whereas the frontal cortexes were collected and fixed in Bouin’s solution (Sigma-Aldrich, St. Louis, MO, USA). Fecal pellets were collected from the colon and preserved at −80 °C for microbiome analysis.