Gb13028

After paraffin sections were rehydrated, slices were incubated in an antigen retrieval solution and blocking serum. Then, the primary antibodies, α-SMA (Servicebio, GB13044, 1:300), CD31 (Servicebio, GB113151, 1: 1,000), transforming growth factor-β₁ (TGF-β₁, Servicebio, GB13028, 1: 200), tumor necrosis factor-α (TNF-α, Servicebio, GB13452, 1: 200), interleukin-4 (IL-4, Bioss, bs-0581r, 1: 200), interferon-γ (IFN-γ, Proteintech, 15365-1-AP, 1: 2000), iNOS (Proteintech, 18985-I-AP, 1: 1,000), and CD206 (Novus, NBP1-90020, 1: 1,000), were added at 4°C overnight. Next, these sections were incubated with HRP-labeled goat anti-rabbit IgG secondary antibody for 50 min at room temperature. Subsequently, the sections were reacted with DAB solution after being washed in PBS, and the nuclei were counterstained with DAPI. Images of smear specimens were also collected by the inverted fluorescence microscope.

USL specimens were obtained from tissues extracted by hysterectomy. Samples (approximately 5 mm³ in size) were taken from the dorsal part of the cervix, 1 cm from the beginning of the cervix (at the left side), and verified by histological analysis.
Tissue samples were fixed in 10% neutral buffered formalin, embedded in paraffin, and treated with citric acid buffer (pH 6.0) in a microwave for antigen retrieval. Sections were then incubated in 3% hydrogen peroxide solution at room temperature in the dark for 25 min to inhibit endogenous peroxidases, then blocked in 3% bovine serum albumin at room temperature for 30 min. Reactions with primary antibodies against CD44 (dilution 1:100; GB13065), MMP-2 (dilution 1:500; GB11130), MMP-9 (dilution 1:500; GB11132-2), and TGF-β (dilution 1:100; GB13028) (all from Servicebio, Wuhan, China) were performed overnight at 4°C. After incubation with horseradish peroxidase-labeled secondary antibodies (Servicebio) at room temperature for 50 min, immune reactivity was detected with diaminobenzidine (K5007, DAKO, Wuhan, China); color development was observed under a microscope. Nuclei were counterstained with Harris hematoxylin. The percentages of positively stained areas were quantified using ImageJ software (version 1.8.0, National Institutes of Health, Bethesda, MD, USA).