Horseradish peroxidase hrp labeled secondary antibody

A total of 10 female BALB/c nude mice (aged 4–6 weeks old and weighing 18–20 g) were acquired from the Vital River Laboratory Animal Center (Beijing, China). The nude mice were randomly allocated to two groups (the NC and shASAP1-IT1 groups), of 5 mice per group, based on their body weight, and reared in a specific pathogen-free environment. Next, 2×10⁶ cells were implanted subcutaneously into the nude mice to form tumors. When the maximum diameter of the tumors reached about 1 cm, the nude mice underwent uniform euthanasia, and the tumor tissues were stripped, photographed, and weighed. Formalin-fixed and paraffin-embedded tissue sections were incubated with TGFBR1 primary antibody (dilution 1:200; Cell Signaling Technology), Ki67 antibody (dilution 1:200; Proteintech), and CD34 (dilution 1:200; Abcam) overnight at 4 ℃ and the Horseradish peroxidase (HRP)-labeled secondary antibodies (dilution 1:2,000; Abcam) were then incubated. Animal experiments were performed under a project license (No. 2019033) granted by the ethics committee of the Fourth Hospital of Hebei Medical University, in compliance with national guidelines for the care and use of animals. A protocol was prepared before the study without registration.

Brain tissue samples were fixed in 10% formalin, processed and embedded in paraffin wax. Tissue sections were cut at a thickness of 4 μm onto glass slides. The sections were dried in a 60°C oven for 1 h, and dewaxed using a conventional xylene method, dehydrated in graded alcohols, incubated in 3% H₂O₂ (Sigma-Aldrich, St. Louis, MO, USA) at 37°C for 30 min, boiled in 0.01 M citrate buffer at 95°C for 20 min, and blocked with normal sheep serum at 37°C for 10 min prior to immunostaining. The tissue sections were incubated with anti-TNFRSF1A primary antibodies (Abcam, Cambridge, MA, USA) at 4°C overnight, washed, and incubated with horseradish peroxidase (HRP)-labeled secondary antibodies (Abcam, Cambridge, MA, USA) at room temperature for 30 min. Following incubation and staining with 3,3′-diaminobenzidine (DAB) and hematoxylin (Sigma-Aldrich, St. Louis, MO, USA), the tissue sections were mounted with a glass coverslip and observed by light microscopy at high magnification (×400) microscope to evaluate the expression of the TNFRSF1A protein.

The total protein of NPMSCs was extracted by a Whole Cell Lysis assay (Keygen Biotech, China), and the protein concentration was determined by a BCA protein assay kit (Beyotime, China). Each sample containing 30 µg of protein was separated by 10%-12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene difluoride membrane (Millipore, United States). After being blocked in 5% skim milk for 2 h at room temperature, the membranes were incubated overnight with the following primary antibodies at 4 °C: P16 (1:1000; Proteintech, China), p21 (1:1000; Proteintech, China), p53 (1:5000; Proteintech, China), SIRT1 (1:1000; ABclonal, China), IL-1β (1:1000; ABclonal, China), IL-6 (1:1000; ABclonal, China), matrix metalloproteinase-13 (MMP-13, 1:1000; ABclonal, China) and Gapdh (1:1000; Servicebio, China). The membranes were then incubated with horseradish peroxidase (HRP)-labeled secondary antibodies (1:5000; Abcam). The protein bands were observed with an enhanced chemiluminescence system, and the expression of protein was analyzed by ImageJ software.

A total of 20 female BALB/c nude mice (aged 4–6 weeks old and weighing 18–20 g) were acquired from the Vital River Laboratory Animal Center (Beijing, China). The nude mice were randomly allocated to 4 groups (si-NC, si-ENST00000534735, pcDNA3.1, pcDNA3.1-ENST00000534735) of 5 mice based on their body weight and reared in a specific-pathogen-free (SPF) environment. 2 × 10⁶ cells were implanted subcutaneously into nude mice to form tumors. When the maximum diameter of tumors reached about 1 cm, the nude mice underwent uniform euthanasia, and the tumor tissues were stripped, photographed, and weighed. After paraffin embedding and sectioning, interleukin (IL)-18 (1:100; Proteintech, Rosemont, IL, USA) and IL-1β (1:500, Proteintech) primary antibodies and horseradish peroxidase (HRP)-labeled secondary antibodies (1:2,000, Abcam, Cambridge, UK) were incubated. The expression levels of IL-18 and IL-1β protein in tissue samples were detected. For the histopathological examination, sections were stained with hematoxylin and eosin (HE). The TdT-mediated biotinylated nick end-labeling (TUNEL) staining procedure followed the manufacturer's instructions before using a light microscope analysis. The Ethics Committee of The Second Hospital of Hebei Medical University approved the study (No. 2022-AE298).

The antibodies in this experiment were obtained from Proteintech. Treated cells were lysed in cell lysis buffer containing 1 mM PMSF (Beyotime Institute of Biotechnology) on ice for 15 min and centrifuged at 16,100 × g for 5 min at 4°C. Next, 50-µg samples of total protein were dispersed on 10% SDS-PAGE gels by electrophoresis and transferred onto cut-out nitrocellulose membranes (Beyotime Institute of Biotechnology). The membranes were blocked for 1 h with 5% skimmed milk at room temperature and incubated with anti-SERPINE1 antibody (cat. no. A00637-1; 1:1,000 dilution; Boster Biological Technology), VEGFA (cat. no. BA0407; 1:500 dilution; Wuhan Boster Biological Technology Co., Ltd.), cleaved caspase-3 (product code ab32042; 1:500 dilution; Abcam), Bax (cat. no. A00183; 1:1,000 dilution; Wuhan Boster Biological Technology Co., Ltd.) and β-actin (product code ab8224; 1:1,000 dilution; Abcam) overnight at 4°C. Subsequently, they were incubated with horseradish peroxidase (HRP)-labeled secondary antibodies (1:1,000 dilution; product code ab7090; Abcam) for 1 h at room temperature. After incubation, the membranes were developed using chemiluminescent substrates (Beyotime Institute of Biotechnology). The densitometric quantification of the bands were calculated using the ImageJ software (version 1.50b; National Institutes of Health; http://imagej.nih.gov/ij/).

bEnd.3 cells were disrupted using Radio Immunoprecipitation Assay (RIPA) lysis buffer (Beyotime) plus 1% protease inhibitor (Invitrogen). Protein concentration was assessed using BCA protein assay kit (Invitrogen). 20 μg protein samples were loaded onto sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel and then electro-transferred onto polyvinylidene fluoride (PVDF) membrane (Bio-Rad, Hercules, CA, USA), which was blocked with 5% skim milk for 1 h. After that, the membrane was incubated with primary antibodies, including anti-B cell leukemia/lymphoma 2 (anti-Bcl-2; ab59348; Abcam, Cambridge, MA, USA), anti-Bcl-2 associated X, apoptosis regulator (anti-Bax; ab182733; Abcam), anti-Cleaved caspase 3 (anti-Cleaved-cas3; ab231289; Abcam), anti-endothelial nitric oxide synthase (anti-eNOs, ab199956; Abcam), anti-EPHA4 (E6900; Sigma, St. Louis, MO, USA) and anti-GAPDH (ab8245; Abcam). After washing three times, horse radish peroxidase (HRP)-labeled secondary antibody (Abcam) was incubated with the membrane for 2 h. Protein signal was measured by the enhanced chemiluminescent visualization (ECL) system (Pierce, Rockford, IL, USA).