Anti tslp

Paraffin sections were stained with HE and then observed by two independent physicians. The number of eosinophils was counted at high power (HP) magnification (400×), and 10 HP fields were randomly selected and analyzed. Referring to the method of Liu et al. [36 (link)], CRSwNP samples were classified as eosinophilic when the percentage of eosinophils exceeded twice the SD of the mean of controls (4.8% + 2×2.12%= 9.04%; therefore, 9% was chosen as the cut-off value).
All monoclonal antibodies (anti-IL-17A, anti-HIF1α, anti-HIF2α, anti-IL-17A receptor, anti-IFN-γ, and anti-TSLP) were purchased from Abcam (Cambridge, MA, USA). An anti-IL-5 polyclonal antibody was purchased from Novus Biologicals. Details of the experimental methods are included in the Supplemental material. Image Pro-Plus 6.0 software was used to analyze images. All images are taken at exactly the same conditions using the same microscope and camera ;image brightness was normalized to background levels. We selected epithelial layer as area of interest (AOI) for measurements. Using Image Pro Plus, the images were changed to grayscale digital images to test total optical density (OD) and calculate the area of AOI; then calculated the mean OD using total OD/area of AOI. The mean OD represents the density of dye staining and reflects the content of the target protein.

Sections were blocked in 5% normal goat serum and incubated with the primary antibodies anti-TSLP (1:100, Abcam, MA, U.S.A.) and anti-phospho-p65 (S536) (1:100, Abcam, MA, U.S.A.), respectively. The sections were then incubated in an appropriate biotinylated immunoglobulin and avidin–biotin peroxidase complex. The negative control was obtained by omitting the primary antibody. Stained sections were observed using a DM4000B Microscope. The results of TSLP and NF-κB staining were estimated as an average of optical density (AOD) using ImagePro-Plus 6.0 software.

Immunohistochemical staining of tissues was performed using an established avidin-biotin detection method (Vectastain ABC kit, Vector Laboratories Inc., Burlingame, CA, United States). The following primary antibodies were used: anti-HDAC3 (1:100, Santa Cruz Biotechnology); anti-MCP1 (1:100, Invitrogen); anti-CD163 (1: 700, Abcam); anti-TSLP (1:500, Abcam); anti-BTG1 (1:200, Abcam), and anti-inducible nitric oxide synthase (iNOS) (1: 500, Santa Cruz Biotechnology). After washing, biotinylated secondary antibody was applied at 1:100 or 1:200 dilutions for 1 h. Color was developed with diaminobenzidine (Vector Laboratories, Inc.). Sections were counterstained with Mayer's hematoxylin. The sections incubated without primary antibody served as controls. To visualize tissue mast cells, the sections were stained with 0.1% toluidine blue (Sigma) in 0.1 N HCl for 15 min.