Diaminobenzidine dab kit

Immunohistochemistry (IHC) stains were used to evaluate the expression of major basic protein (MBP) and tryptase, which are markers of eosinophil and mast cell degranulation, respectively26 (link). After dewaxing and rehydrating, tissue sections (D1, D2) were treated with 0.3% hydrogen peroxide in methanol to block endogenous peroxidase activity followed by pepsin or heat-mediated antigen retrieval. After blocking with 3% goat serum for 20 minutes at room temperature to minimize nonspecific staining, the sections were incubated with mouse anti-eosinophil MBP antibody (1:50, AbD Serotec, UK) and mouse anti-mast cell tryptase antibody (1:26000, Abcam, USA) for 1 hour at room temperature. After washing, the sections were treated with HRP-labeled goat anti-mouse IgG (ZSGB, China) for 30 minutes. The reaction was visualized using diaminobenzidine (DAB kit, ZSGB, China) and hematoxylin staining. Eosinophil degranulation positive was defined by the presence of MBP granules stain in randomly selected fields. If there was evidence of eosinophil degranulation on IHC stain, five non-overlapping fields on the slides (400× magnification, Nikon) were randomly selected and analyzed by two independent observers (LD, LM), to determine the mean numbers of degranulated eosinophil and mast cell and expressed per HPF.

The human endometrioid endometrial carcinoma cells (KLE, low differentiation) were purchased from China Center for Type Culture Collection (Wuhan, China) and cultured in DMEM/F12 1:1 (HyClone Corporation, Beijing, China) containing 10 % fetal bovine serum (Gibco, CA, USA), 100 U/ml of penicillin and 100 U/ml of streptomycin. The cells were seeded in 24-well plates with cover slips at 5 × 10⁴ cells/well at 37 °C in 95 % air and 5 % CO₂. After the glandular epithelial cells, stromal cells and KLE cells were cultured in 24-well plates with cover slips overnight, the cells on the cover slip were fixed with 4 % paraformaldehyde for 30 min. After successively endogenous peroxidase blocking and 10 % goat serum blocking, the cells were respectively incubated with anti-human antibodies against vimentin (1:100) (ZSJQB Co., Ltd. Beijing, China), cytokeratin (1:100) (ZSJQB Co., Ltd. Beijing, China) and PDCD5 (1:1000) (abcam, Shanghai, China) overnight at 4 °C. The cells were washed with PBS and incubated with horseradish peroxidase-conjugated secondary antibodies (ZSJQB Co., Ltd. Beijing, China) for 30 min at 37 °C. Then the protein expression was visualized using diaminobenzidine (DAB) kit (Zsbio, Beijing, China) and the nuclei were counterstained with hematoxylin. Each sample was performed in duplicate.

The materials are as follows: recombinant TNF-α (PeproTech, NJ, USA); ABT-199, SP600125, and lenalidomide (MedChemExpress, NJ, USA); TNF-α, myeloperoxidase (MPO), interleukin-1 β (IL-1β), IL-6, IL-8, and IL-18 enzyme linked immunosorbent assay (ELISA) kits (BD Biosciences, CA, USA); rabbit anti-mouse FoxO3a antibody, rabbit anti-mouse Ly6G antibody, rabbit anti-mouse JNK antibody, rabbit anti-mouse Bcl-2 antibody, rabbit anti-mouse glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody, horseradish peroxidase- (HRP-) labeled goat anti-rabbit secondary antibody (Abcam, Cambridge, UK); HRP-labeled anti-mouse secondary antibody, bicinchoninic acid (BCA) Protein Assay Kit (Beyotime Biotechnology, Shanghai, China); Naphthol AS-D Chloroacetate (Specific Esterase) Kit (Sigma, NY, USA); Diaminobenzidine DAB Kit (ZLI9019; ZSGB-BIO); Dulbecco's modified Eagle's medium (DMEM) medium, trypsin, and fetal bovine serum (FBS) (Gibco, NY, USA); FoxO3a small interfering ribonucleic acid (siRNA) (Guangzhou RiboBio Co., Ltd., Guangzhou, China); and Cell counting kit-8 (CCK-8) and Annexin V-fluorescein isothiocyanate/propidium iodide (FITC/PI) Apoptosis Assay Kit (Jiangsu KeyGEN BioTECH Corp., Ltd., Jiangsu, China).

The slides were dried, dewaxed, rehydrated, and treated with 3% hydrogen peroxide (H₂O₂) in methanol for 10 min. Antigen retrieval was performed by boiling the slides twice in a sodium citrate buffer (0.01 mol/L, pH 6.0). A 5% bovine serum albumin (BSA; Boster Biological Technology Co. Ltd, Wuhan, China) was used in a 1:10 dilution during a 30 min incubation period at 37 °C. There was a two-hour incubation period with the Ki67 antibody (Abcam, ab15580; 1:800 dilution) at 37 °C. Sections were treated with a goat anti-rabbit IgG secondary antibody (ZSGB-BIO, Beijing, China) for one hour at 37 °C. With the exception of the blocking step, at every step, washing was completed thrice in PBS for 5 min. Positive cells were observed with a diaminobenzidine (DAB) Kit (ZSGB-BIO, Beijing, China), stained with hematoxylin, and made into permanent pieces. A total of 15 microscopic fields per sample were captured using a light microscope under 20× magnification (Leica DM3000, Leica Microsystems, Wetzlar, Germany). The number of Ki67-positive cells, at least 30 well-oriented complete crypts per sample was counted manually for analysis [24 (link)].