Serum cytokines ELISA assay
The serum levels of IL-6, IL-21, IL-23, and TGF-β
were
quantified using commercially available ELISA kits according to the
manufacturer’s instructions (Proteintach Group Inc, Chicago, USA).
Immunohistochemical
double staining analysis of Th17 and Tregs in thymoma
A two-color immunohistochemical double stain with CD4 and IL-17A primary
antibodies (Th17 cells) and CD4 and FOXP3 primary antibodies (Tregs) was
performed according to the manufacturer’s protocol (KIT-9999: Fuzhou
Maixin Biotechnology Development Co., Ltd. China). Paraffin-embedded
thymoma tissue sections (4 µm) were de-waxed in xylene, dehydrated in
ethanol, and subjected to antigen retrieval with Tris-EDTA under high
temperature and high pressure for 3 min. Endogenous peroxidase activity
was blocked by incubation with 3% H2O2for 10 min. Non-specific reactions were blocked by incubation with
serum-free medium for 10 min at room temperature. Two sections from the
same sample were incubated with anti-IL-17A antibody (1:100 dilution;
ab136668, Abcam, Cambridge, UK) or anti-FOXP3 antibody (1:200 dilution;
ab54501, Abcam) overnight at 4 °C. The following morning, the sections
were incubated with goat anti-rabbit secondary antibody, and then were
stained with BCIP/NBT (mandarin blue indicated positive staining). The
slides were rinsed in PBS and incubated with double staining intensifier
and serum-free medium for 10 min. The slides were then incubated with
the second primary antibody, anti-CD4 (1:100 dilution; ab846, Abcam),
overnight at 4 °C. Then, the sections were incubated with goat
anti-rabbit secondary antibody and stained with AEC
(3-amino-9-ethylcarbazole chromogen) solution and hematoxylin. Thus, the
two primary antibodies could be easily differentiated by the various
chromogens (IL-17A, mandarin blue immunolabeling in cytoplasm; FOXP3,
mandarin blue immunolabeling in cytoplasm or nuclei; CD4, red
immunolabeling in cell membrane), as well as by cellular stain
localization. Double-positive cells were counted on five adjacent
high-power fields (×400) of each section.
Analysis of
RORγt and FOXP3 in
thymoma
The immunohistochemical procedure of RORγt and FOXP3 staining as
described above was used for staining of thymoma, with the exception of
the second primary antibody. Sections were incubated with anti-human
RORγt antibody (1:40 dilution; ab219496, Abcam) and anti-human FOXP3
antibody (1:50 dilution; ab54501, Abcam). The sections were then
incubated with goat anti-rabbit secondary antibody and stained with DAB
and hematoxylin. Brown granules in the nuclei were considered to
represent a positive signal. The frequency of positive cells in five
high-power fields (magnification, ×400, Leica DMIL, Germany) of each
section was determined. More than 10% positive cells in each high-power
field were found to have positive expression.
Real-time quantitative PCR (RT-qPCR) analysis of RORC
(RORγt) and FOXP3 mRNA