Hrp conjugated anti rabbit igg

Primary antibody against B cell leukemia/lymphoma 2 (Bcl-2) was obtained from Boster (Wuhan, Hubei, China). Primary antibodies against polyclonal antibodies against proliferating cell nuclear antigen (PCNA), Bcl-2 associated X (Bax), survivin, matrix metallopeptidase 9 (MMP9), matrix metallopeptidase 2 (MMP2), β-Actin and normal rabbit or mouse immunoglobulin G (IgG) were obtained from Proteintech (Wuhan, Hubei, China). Primary antibody against Notch4 was obtained from Santa Cruz Biotechnology (Dallas, TX, United States). Primary antibodies against P38/p-P38 MAPK, JNK/p-JNK, ERK/p-ERK were obtained from Cell Signaling Technology (Danvers, MA, United States). HRP-conjugated anti-Rabbit IgG and HRP-conjugated anti-mouse IgG were obtained from Servicebio (Wuhan, Hubei, China). HRP-conjugated anti-Rabbit IgG light chain and HRP-conjugated anti-mouse IgG light chain were obtained from Abbkine Scientific (Redlands, CA, United States). Protein G magnetic beads were obtained from Cell Signaling Technology (Danvers, MA, United States). U0126, SP600125 and SB203580 were obtained from MedChemExpress (Monmouth Junction, NJ, United States).

The femur samples were fixed with 4% PFA for 2 days and then embedded in paraffin after two weeks of decalcification with 10% EDTA treatment. Serial 5 μm sections of the femur were made using a microtome, and the femur sections were stained with immunohistochemical staining. Briefly, femur sections were processed for antigen retrieval for 15 min and blocked in 3% BSA for 30 min. The sections were then incubated overnight with primary antibody against osteocalcin (1:2000, Servicebio, China) at 4 °C. After three washes with PBS, the sections were incubated with HRP-conjugated anti-rabbit IgG (1:400, Servicebio, China) for 50 min. A 3N-diaminobenzidine tetrahydrochloride (DAB) kit (Servicebio, China) was used to detect immunoactivity, followed by counterstaining with hematoxylin (Servicebio, China). The sections were examined under a fluorescence microscope (Nikon, Japan).

For immunohistochemistry, tumor and non-tumor tissues from CRC patients after surgery were rinsed with PBS and fixed with 10% formalin. After fixation, tissues were dehydrated in ethanol overnight for paraffin embedding. After paraffin embedding, the paraffin block was cut into 5 μm slices. After deparaffinization, the tissue slices were rehydrated in 3,3′-diaminobenzidine solution and were treated with H₂O₂ to close the endogenous peroxidase. Then the slices were incubated in retrieval solution at 60 °C overnight for heat-induced epitope retrieval, followed by treatment with 1% BSA for 2 h to block non-specific antibody binding. Then, the slices were incubated with 1 μg/ml of primary antibody against human Dectin-1 (Cat# ab140039, Rabbit polyclonal to human and Rat Dectin-1, Abcam Inc, Cambridge, UK) overnight at 4°C, followed by HRP-conjugated anti-rabbit IgG (Cat# GB23303, Servicebio co. Wuhan, China) for 15 min at room temperature. Tissue slices were then treated with DAB substrate solution for 1-3 min, and with hematoxylin counterstain for additional 10 seconds at room temperature. Photos of IHC were taken by Leica DM6FS with imaging scanning system (Leica Camera AG, Wetzlar, Germany).