Biotinylated secondary antibody

Paraffin sections were baked in an oven at 65 °C for 2 h. After dewaxing with xylene and dehydrating with gradient alcohol, antigen retrieval was performed by immersing sections in citric acid solution at 100 °C for 15 min. Then sections were cooled to room temperature and washed twice with PBS. Sections were treated with 3% hydrogen peroxide and incubated at 37 °C for 10 minutes, washed 3 times with PBS, and blocked with normal goat serum. The sections were then incubated overnight at 4 °C with p-mTOR antibody (working concentration 1: 100). Tissue slices were washed with PBS three times, incubated with biotinylated secondary antibody (Zhongshan Golden Bridge Biotechnology Company, Beijing China) for 30 min at room temperature. After another PBS wash, sections were incubated for 30 min with streptavidin-peroxidase complex (Zhongshan Golden Bridge Biotechnology Company, Beijing China). The color reaction was developed with diaminobenzidine, finally, sections were counterstained with hematoxylin. Tumors with > 25% p-mTOR positive cells were deemed positive for antigen expression. Pathological diagnoses of tissue sections were determined by two experienced pathologists blinded to treatment.

The minimal change disease (MCD) patients confirmed by renal biopsy were used as negative controls. Paraffin-embedded kidney biopsies were cut in 3 micrometer thick sections placed on slides, heat, deparaffinized by xylene, hydrated by gradient descent alcohol, recovered by microwave heating in citrate buffer (pH 6.0), and incubated in 0.3% hydrogen peroxide phosphate-buffered saline to inactivate endogenous peroxidase. Next, sections were stained with rabbit polyclonal antibodies for von Willebrand factor (VWF) (1:1200 dilution, Abcam, Cambridge, UK) overnight at 4°C in a moist chamber. Biotinylated secondary antibodies (1:100 dilution; Zhongshan Golden Bridge, Beijing, China) were incubated for 20 min at 37°C and an avidin-biotin-peroxidase complex (1:20 dilution; Zhongshan Golden Bridge, Beijing, China) was applied. Finally, diaminobenzidine was used as chromogen and hematoxylin as counterstaining. The staining for VWF was observed and assessed semi-quantitatively.

Tissue sections for immunohistochemical staining were dewaxed, incubated with 3% H₂O₂, and heated with citrate buffer (pH 6.0). After being blocked by goat serum, sections were incubated overnight with CD31 primary antibody (Abcam, UK) at 4 °C. The sections were then incubated for 1 h with biotinylated secondary antibodies (Zhong Shan Golden Bridge Biotechnology, China). Peroxidase activity was detected by diaminobenzidine (DAB). Finally, sections were counterstained with hematoxylin.

Immunohistochemistry (IHC) was performed on all tissue samples using biotin-streptavidin HRP detection systems. After deparaffinization with xylene and dehydration in a graded alcohol series, the tissue sections were subjected to antigen retrieval by microwaving in sodium citrate buffer for 10 min and then inhibiting endogenous peroxidase activity. After nonspecific binding was blocked, the slides were incubated with YAP (1:200) and β-catenin (1:200) antibody (Santa Cruze, USA), Ki67 (1:300, MXB biotechnologies, China) in phosphate-buffered saline (PBS) overnight at 4 °C in a humidified container. Biotinylated secondary antibodies (Zhongshan Golden Bridge Biotechnology, China) were then used according to the manufacturer’s recommendations. The sections were incubated with HRP-streptavidin conjugates appropriate for detecting these proteins. The brown color indicative of peroxidase activity was developed by incubation with 0.1% 3,3′-diaminobenzidine (Zhongshan Golden Bridge Biotechnology) in PBS with 0.05% H₂O₂ for 5 min at room temperature. The appropriate positive and negative controls were included in each run of IHC.

Immunohistochemical studies were performed as previously reported [15 (link)]. Normal brain tissue was obtained from a frontal lobe trauma patients. Deep gray matter in frontal lobe was used. The sections were incubated with a monoclonal antibody specific for NCL (Santa Cruz, CA) at a 1:100 dilution overnight at 4°C for 24h, and then detected using biotinylated secondary antibodies (Zhongshan Golden Bridge Biotechnology Ltd. Co., China) based on the manufacturer’s protocols. The staining of the slides was carried out by the HRP-streptavidin conjugates. The slides were visualized with diaminobenzidine, and then counterstained with hematoxylin. PBS was used instead of the primary antibodies for the negative controls.

Immunohistochemistry (IHC) was performed on all colon cancer samples and the tissues of xenograft tumour using biotin-streptavidin HRP detection systems. Paraffin-embedded tissue sections were collected. After deparaffinization with xylene and dehydration in a graded alcohol series, the tissue sections were subjected to antigen retrieval by microwaving in sodium citrate buffer for 10 min and then inhibiting endogenous peroxidase activity. After nonspecific binding was blocked, the slides were incubated with ERRα (1:100) and IDH3A (1:200) antibody (Santa Cruz Biotechnology, CA, USA); c-Myc and Cyclin D1 antibody (1:200; Abcam, Cambridge, UK,) in phosphate-buffered saline (PBS) overnight at 4 °C in a humidified container. Biotinylated secondary antibodies (Zhongshan Golden Bridge Biotechnology Co. Ltd., China) were then used according to the manufacturer’s recommendations. The sections were incubated with HRP-streptavidin conjugates appropriate for detecting ERRα; IDH3A; c-Myc and Cyclin D1. The brown color indicative of peroxidase activity was developed by incubation with 0.1% 3,3-diaminobenzidine (Zhongshan Golden Bridge Biotechnology Co. Ltd. China) in distilled water for 1–3 min at room temperature. The appropriate positive and negative controls were included in each IHC assay.