Hoechst staining

After deparaffinization, rehydration and proteinase K-induced antigen retrieval, nasal polyp sections were blocked and stained with anti-EpCAM (Cell Signaling Technology, Danvers, MA, USA) and CCL4 (Bioss, Woburn, MA, USA), followed by incubation with Alexa 488 goat anti-rabbit and Alexa 647 goat anti-mouse antibodies (Jackson, PA, USA). After co-incubation overnight with purified eosinophils, BEAS-2B cells were fixed with 4% formaldehyde for 20 min, permeabilized, and blocked. Cells were then incubated with a mouse monoclonal antibody to CCR5 (Abcam, Cambridge, UK), followed by CF 647-labeled goat anti-mouse antibody incubation (Biotium, Fremont, CA, USA). Slides were visualized using a FV3000 confocal microscope (Olympus, Tokyo, Japan) with the identical settings. Control antibodies and Hoechst staining (Invitrogen, Paisley, UK) were included in each experiment. To evaluate the ratio intensity of CCL4 to EpCAM, ImageJ was used [14 (link)].

Both WT and Fib-3 KO CSs were fixed with 4% paraformaldehyde for an hour at room temperature to stop the cell distribution process, permeabilized in PBS/0.01% sodium azide (PBSA) containing 0.02% Triton-X-100 for 60 min, blocked with a 3% bovine serum albumin/PBSA solution, and then incubated with appropriate primary (15 μg/mL) and secondary (10 μg/mL) antibodies at 4 °C (18 h). For cell behavior analysis, fixed CSs were exposed to primary antibodies against cardiac troponin T and CD31, to stain CMs and ECs, respectively (1 (link)). Nuclei were stained with Hoechst staining (Invitrogen, Carlsbad, CA). CSs were mounted on a glass slide using Vectashield Mounting Medium (Vector Laboratories, Burlingame, CA). Fluorescent imaging used a Zeiss LSM 800 Laser Confocal Microscope (Carl Zeiss AG, Oberkochen, Germany). Optical sectioning along the Z axis was performed, and the images collapsed into a single focal plane using the manufacturer's software. Images were processed using Adobe Photoshop CC (Adobe Systems, Inc., San Jose, CA). 3D rendering analysis was performed on the confocal images using Imaris software which allows conversion of each pixel of a z-stack into a 3D voxel (3D sectioning) to obtain quantitative information from the image (1 (link), 25 (link)).

Nasal polyps were formalin-fixed and paraffin-embedded following endoscopic sinus surgery under general anesthesia. After deparaffinization, rehydration, and proteinase K-induced antigen retrieval, sections were blocked and stained with an epithelial cell marker anti-EpCAM (Cell signaling Technology, Danvers, MA, USA), an eosinophil marker MBP (Bio-Rad, Hercules, CA, USA), and CCL4 (Bioss, Woburn, MA, USA), followed by Alexa 488 goat anti-rabbit antibody and Alexa 647 goat anti-mouse antibody (Jackson, PA, USA). The intensity ratio of CCL4 to EpCAM was calculated using ImageJ 11.24.13. Briefly, all CCL4 or EpCAM signals were labeled, and the intensity was evaluated using ImageJ (a single signal was reversed to 8-bit). BEAS-2B cells coincubated with purified eosinophils or eosinophils were fixed with 4% paraformaldehyde, permeabilized, and, subsequently, blocked. The cells were then incubated with the primary antibodies, and CCL4 (Bioss), CCR5 (Abcam), PDGFRβ (Cell signaling Technology, Woburn, MA, USA), and Src (Bioss) were evaluated, as previously described. Additionally, control antibodies and Hoechst staining (Invitrogen, Paisley, UK) were included in each experiment, and slides were visualized using FV3000 confocal microscopes (Olympus, Tokyo, Japan).