Anti tnf α

Histopathological analysis and immunohistochemistry were carried out as described [18] (link). Anti-p65 (Santa Cruz Biotechnology, Santa Cruz, CA, USA; 1∶200), anti-p-IKKα/β (Cell Signaling Technology, Danvers, MA, USA; 1∶100), anti-p-S6(S235/236) (Cell Signaling Technology; 1∶400), anti-AKT (Abcam, Cambridge, MA, USA; 1∶100), anti-p-AKT(S473) (Abcam; 1∶100); anti-Bax (Epitomics, Burlingame, CA, USA; 1∶300), anti-Ki67 (Bioss, Beijing, China; 1∶50), anti-IL-1β (Abzoom, Dallas, USA; 1∶200), anti-IL-6 (Sanying, Wuhan, China; 1∶300), and anti-TNF-α (Boster, Wuhan, China, 1∶150) were used in the immunohistochemical studies. Secondary antibody was diluted 1∶100 and applied for 2 h at room temperature. The Vectastain ABC Elite System (Vector Laboratories, Burlingame, CA) was used to visualize staining for IHC. The immunostained slides were observed under a microscope. Images were taken by a digital camera and semi-quantity of the signal was analyzed by counting mean density of the immunoreactivities against all primary antibodies. Brown or yellow was regarded as positive signal. Image data were analyzed with NIS-Elements AR 3.0 software (Nikon, Japan). Immune cell, apoptosis, and proliferation indices were generated by counting the number of positive cells per high-powered field (HPF; 40× objective) within each mouse [19] (link).

Immunofluorescence staining (6 villous and decidual tissues including 3 controls and 3 RM women) was used to localize and compare the distribution of CYP27B1 and TNF-α, IL-2, IFN-γ and IL-10. The 5-μm-thick cryosections were air-dried and immersed in a buffer (30% H₂O₂: distilled water = 1:10) for 10 min at room temperature. After blocking with normal goat serum for 20 min at room temperature, the sections were incubated with anti-CYP27B1 polyclonal antibody diluted at 1:100 for overnight at 4°C, followed by incubation with antibody including diluting anti-TNF-α (1:500), IL-2 (1:50), IFN-γ (1:500), and IL-10 (1:100) (Boster Biological Technology, Ltd., China) for 10 h at 4°C, respectively. Then, the sections were incubated with carbocyanine 3 (Cy3)-conjugated and AlexaFlur 488-conjugated secondary antibody (Boster Biological Technology, Ltd., China) for 2 h at 37°C. The omitted primary antibody was used as negative control. Confocal laser scanning microscopy (CLSM) (TCS SP2, Leica Co., Germany) was used for evaluation of morphology and the level of protein expression. To further exclude operator bias, observations were performed on coded samples in a blinded fashion. The fluorescence excitation was provided by a 488/560 nm argon laser beam and emission was 535/650 nm for FITC/Cy3. The images were analyzed using the software Image Plus (Leica, Germany).

The procedure for protein extraction was the same as in a previous study (Yi et al., 2018 (link)). Protein samples of 20–30 μg were subject to electrophoresis on SDS polyacrylamide gels. After transferring the proteins to membranes, 5% skimmed milk was used to block at room temperature for 2 h, followed by incubating at 4°C overnight with primary antibodies: anti-P2X7 (Alomone Labs, Jerusalem, Israel), anti-TACE (Novus Biologicals Co., Littleton, United States), anti-TNF-α (Boster Biological Technology, Wuhan, China), anti-NF-κB (Affinity Biosciences, Ohio, United States), anti-STAT3 (Cell Signaling Technology, Beverly, MA, United States), anti-phosphorylated (p)-STAT3 (Cell Signaling Technology, Beverly, MA, United States) or anti-β-actin (ZSGB-BIO, Beijing, China). After washing with TBST for 3 × 10 min, the membranes were incubated at room temperature for 2 h with the second antibodies: goat anti-rabbit IgG (Proteintech, Rosemont, United States), or goat anti-mouse IgG (Proteintech, Rosemont, United States). After 10 min wash with TBST thrice, the membranes were exposed and developed in a gel imaging system. Image-ProPlus 6.0 was used to analyze the results.