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.