Ds fi2 camera

FFPE lung sections were deparaffinized and incubated during 1 h in 1% Alcian Blue 8GX solution in 3% acetic acid (Sigma). A counterstaining was performed using Nuclear Fast Red solution (Sigma) during 15 min. Microscopy was performed using a Nikon DS Fi2 camera and NIS-Elements software (Nikon). Positive cells for Alcian Blue staining (AB⁺) were manually counted in the main bronchi (Fig. S2) and results were expressed as cells/mm epithelium.

Myogel was thawed and diluted to 0.5 mg/mL and mixed with 0.5 mg/mL type 1 rat-tail Collagen (Corning, NYC, NY, USA). One hundred µL of media was removed, and 100 µL Myogel/Collagen1 mixture was gently dispensed into the inner wall of each well of the ULA-plate containing 4-day old spheroids. The gel was allowed to solidify for 30 min, and then another 100 µL of fresh medium was added. Similar to the invasion assay, gel and media were prepared with and without IL-17F at 10, 50, or 100 ng/mL. HSC-3 or SCC-25 alone or in combination with CAFs were exposed to various concentrations of IL-17F in the Myogel/Collagen mixture for 5 subsequent days. Spheroid images were taken using an eclipse TS100 inverted microscope (Nikon DS-Fi2 camera; Nikon, Tokyo, Japan). Cancer cell invasion was calculated as the distance from the farthest cancer cell to the center of the tumor spheroid.

IHC was performed using the previously described protocol for paraffin sections.²⁵ After deparaffinization, endogenous peroxidase activity was inhibited by incubation with 3% hydrogen peroxide for 10 min and samples were washed with PBS. Heat‐induced antigen retrieval was performed by incubation in citrate buffer and the sections were incubated in a blocking solution for 10 min to prevent non‐specific binding. After the blocking step, all sections were incubated overnight with an anti‐tumour necrosis factor‐alpha (TNF‐α) (E‐AB‐52065, 1:150, Elabscience) and anti‐cyclooxygenase‐2 (anti‐COX‐2) primer antibodies (E‐AB‐17010, 1:150, Elabscience). The next day, biotinylated antibodies (TP‐125‐BN, Thermo Scientific) and streptavidin peroxidase (TS‐125‐HR) were applied to the sections. Then, a 3,3′‐Diaminobenzidine substrate kit (ab64238, Abcam) was performed for 3–5 min. Finally, the sections were counterstained with Mayer's haematoxylin and visualized using Nikon Eclipse Ni microscope with a Nikon DS‐Fi2 camera attachment. Images were imported into ImageJ (Fiji) for quantification of percent area of TNF‐α and COX‐2 expression analyses. TNF‐α and COX‐2 intensity (% Area) measurements in the ileum and colon were performed over 10 random areas of interest (3 different sections for each rat per group).²⁶