Sodium citrate buffer

Binding assay was performed as described previously (54 (link)). Briefly, cecum tissue samples from 2-week-old chickens without coccidian infection were dehydrated, waxed, and fixed in 4% paraformaldehyde for preparation of tissue sections. Tissue sections were boiled (about 10 min) in sodium citrate buffer (Beijing Solarbio Science & Technology Co., Ltd.) to repair the antigen. The sections were blocked in 5% bovine serum albumin in TBST for 2 h. After cooling to room temperature, sections were incubated with rEtMIC8 protein overnight at 4°C. Samples incubated with PBS were used as the controls. Subsequently, sections were sequentially incubated with anti-EtMIC8 MAb for 1 h at 37°C, the FITC-conjugated secondary antibody for 30 min at 37°C, and DAPI (4′,6-diamidino-2-phenylindole; Beijing Solarbio Science & Technology Co., Ltd.) for 5 min. The tissue sections were detected by fluorescence microscopy (Nikon-ECLIPSE; Japan). The EtMIC8 binding ability that was proportional to the fluorescence intensity was measured by detecting fluorescence intensities of different groups.

Immunohistochemical staining was conducted following established procedures. Briefly, 4-μm sections of bone tissues were deparaffinized in xylene and rehydrated in gradient ethanol. The endogenous antigen was retrieved by incubating slides in 10 mM sodium citrate buffer (Cat #: C1010, Solarbio, Beijing, China) overnight in an oven at 65°C. After antigen retrieving, tissue sections were incubated with 3% hydrogen peroxide at room temperature for 10 min to block the endogenous catalase activity. They were then incubated in a blocking solution (0.1% albumin bovine V mixed with 10% goat serum) for 40 min, and a solution containing a primary antibody (MMP9, 1:1000, Cat #: ab76003, Abcam, Cambridge, UK; MYBL2, 1:100, Cat #: ab76009, Abcam) for 1.5 h. After rinsing with PBS for 3 times, slides were incubated with the secondary antibody solution for 15 min and then with the DAB solution using the MaxVision II HRP kit (Cat #: KIT-5920, MXB Biotechnologies, Fuzhou, China). Nuclei were counterstained with hematoxylin for 2 min. Slides were then dehydrated with gradient ethanol (75–100%) and xylene and mounted with coverslips using the quick-hardening mounting medium. All slides were scanned using an Aperio VERSA 8 Scanner System (Leica, Wetzlar, Germany) and analyzed using the Image J software (Image J 1.48v, NIH, Bethesda, MD, USA).

IHC assay was performed to examine protein expression in human prostate cancer tissue microarray and tumor tissue section from xenografts of prostate cancer models. The sections were dewaxed with xylene and hydrated by a standard xylene-ethanol procedure, followed by antigen retrieval in 10 mM sodium citrate buffer (Solarbio). Endogenous peroxidase activity was blocked with 3% H₂O₂ for 10 min at room temperature. The sections were blocked with normal goat serum to block non-specific sites for 18 min at room temperature. Subsequently, the sections were incubated with the indicated primary antibodies at 4 °C overnight. HRP-conjugated secondary antibody was incubated with the sections at room temperature for 30 min. DAB (Service, Wuhan, China) was applied to visualize the protein expression followed by counterstaining nuclei with hematoxylin. For histomorphometric analysis, tissue sections were stained with hematoxylin and eosin. The sections were captured by a NIS-element imaging system (Nikon, Japan).

Acrylamide (AM) was supplied by Macklin (Shanghai, China). Potassium persulfate, trichloromethane and isopropyl alcohol were purchased from Sinopharm Chemical Reagent (Shanghai, China). Potassium sucrose octasulfate was purchased from Avito (Shanghai, China). Polyvinyl alcohol (PVA), methyl methacrylate (MMA), Span-80, divinylbenzene (DVB) and all other chemicals were purchased from Aladdin (Shanghai, China). Cell counting kit 8 was purchased from Biosharp (Wuhan, China). Live/dead staining kits, sodium citrate buffer and streptozotocin were purchased from Solarbio (Beijing, China). The activated partial thromboplastin time assay kit and prothrombin time assay kit were purchased from Rayto (Shenzhen, China). The 5-ethynyl-2’-deoxyuridine (EdU) kit was provided by Beyotime (Shanghai, China). The MMP-9 protein reagent for the in vitro cell assay was purchased from Novoprotein (Suzhou, China). The transforming growth factor-β (TGF-β) antibody was provided by Boster (Wuhan, China). The vascular endothelial growth factor (VEGF) antibody and epidermal growth factor (EGF) antibody were purchased from ABclonal (Wuhan, China). The MMP-9 antibody was purchased from Proteintech (Wuhan, China). The platelet endothelial cell adhesion molecule-1 (CD31) antibody was provided by Abcam (Shanghai, China).

The testes of mice were fixed with 4% paraformaldehyde (PFA) overnight at 4 °C. Paraffin embedding and sectioning were then conducted using the usual procedures. The slices were dewaxed and rehydrated, immersed in 0.01% sodium citrate buffer (pH 6.0, Solarbio, Beijing, China), and boiled in water for 20 min. Next, the slices were permeated with 0.2% TrionX-100 at room temperature for 10 min. After being washed with phosphate-buffered saline (PBS), the samples were blocked with 5% goat serum at 37 °C for 30 min. Further, the sample was incubated with the primary antibodies at 4 °C overnight. The secondary antibodies conjugated to fluorescein isothiocyanate (FITC, 1:200; Invitrogen, USA) or tetramethylrhodamine isothiocyanate Fluor (TRITC, 1:200; Invitrogen, USA) were used to test the primary antibody. 4′,6-diamino-2-phenylindole (DAPI; Abcam, Cambridge, UK) was used to stain the nuclei. The samples were observed under the LSM900 confocal microscope system (Carl Zeiss AG, Jena, Germany), and the images were captured. The following antibodies were used: anti-γH2AX (1:200; Abcam, Cambridge, UK), anti-DEAD-box helicase 4 (DDX4) (1:200; Abcam, Cambridge, UK), anti-proliferating cell nuclear antigen (PCNA) (1:50; Santa, Texas, USA), anti-GSS (1:200; Affinity Biosciences, Jiangsu, China) antibodies.

An immunohistochemistry assay was performed to detect KIAA1217 expression in the TMA tissue samples. The TMA slide was dewaxed in xylene and rehydrated with a series of graded alcohol solutions. Then, antigen retrieval was performed with sodium citrate buffer (C1032, Solarbio, Beijing, China). According to the protocols of the two-step detection kit (PV-9001, ZSGB-BIO, Beijing, China), the TMA slide was incubated in an appropriate endogenous peroxidase blocker for 10 min at room temperature, followed by incubation with anti-SKT (KIAA1217 is also known as SKT) antibodies overnight at 4 °C and subsequent incubation with response enhancer and enhanced enzyme-labeled goat anti-rabbit IgG polymer for 20 min at room temperature. A DAB Chromogenic Kit (ZLI-9017, ZSGB-BIO, Beijing, China) was used to detect antibody binding, and the reaction was stopped by immersing the TMAs in running water once a brown color appeared. Finally, the TMA slide was counterstained with hematoxylin (G1121, Solarbio, Beijing, China), dehydrated using a series of graded alcohol solutions, and mounted. Images were photographed with an inverted microscope. Appropriate positive and negative controls were included for each run of the IHC assay. The antibodies used in this study are listed in Supplementary Table S3.