Immunohistochemistry (IHC) analysis
For expression analysis of CD40, CD40L, ICOS, ICOSL, formalin-fixed and paraffin-embedded nasal biopsies were cut into 4 μm thick sections deparaffinized by serial treatment. Deparaffinized sections were subjected to antigen retrieval by heating the sections in sodium citrate buffer, pH 6.0. After blocking the endogenous peroxidase in 3% hydrogen peroxide and with 3% bovine serum albumin, the sections were incubated overnight at 4°C in the presence of rabbit-derived primary antibodies against CD40 (1:100, Affinity Biosciences, AF5336), CD40L (1:200, Abcam, Cambridge, MA, USA, ab65854), ICOS (1:500, Abcam, Cambridge, MA, USA, ab224644) and ICOSL (1:200, Abcam, Cambridge, MA, USA, ab233151). Thereafter, each section was incubated with HRP (Horse Raddish Peroxidase) conjugated anti-rabbit secondary antibody (1:500) for 50 min. After washing, sections were incubated with DAB (3,3’-diaminobenzidine tetrahydrochloride) and immediately washed under tap water after color development. Then, sections were counterstained with hematoxylin and mounted with DPX (dibutyl phthalate xylene). The sections were blindly examined with no awareness of the clinical data with an Olympus CX40 Microscope (Olympus Optical Co, Hamburg, Germany). The number of positive cells was counted in 5 random HPFs (x200) and averaged.
For further analysis of co-location of CD40 and ICOSL with eosinophils, immunofluorescence was performed using TSA (Tyramide signal amplification) technique. Sections were deparaffinized and antigen retrieval was performed in Tris-EDTA (Ethylenediaminetetraacetic acid) buffer, pH=9.0. After blocking the endogenous peroxidase, sections were incubated overnight at 4°C in the presence of primary antibody against PRG2 (1:1000, Abcam, Cambridge, MA, USA, ab236851), which is MBP, major basic protein, the predominant constituent of the crystalline core of the eosinophil granule. Then, HRP conjugated anti-rabbit secondary antibody (1:500) was incubated with sections for 50 min at room temperature. Sections were then incubated with 488-TSA at room temperature for 10 min. Next, antigen retrieval was performed before incubating with primary antibody against CD40 (1:250, Affinity Biosciences, AF5336) or ICOSL (1:200, Abcam, Cambridge, MA, USA, ab233151). After washing, sections were incubated with CY3 conjugated anti-rabbit secondary antibody (1:300). The DNA dye 4′,6-diamidino-2-phenylindole (DAPI) was used to visualize the nucleus. Results were captured under fluorescence microscope. Agents not mentioned specifically obtained from Servicebio technology CO, Wuhan, China.