Horseradish peroxidase conjugated swine anti rabbit igg

Paraffin sections of 5 μm thickness were prepared for alkaline phosphatase (ALP), PRX1 and PRX5 immune labeling. Briefly, following xylene treatment, dewaxed paraffin sections were pretreated with 0.3% hydrogen peroxide for 30 min, and then blocked with 1% bovine serum albumin (BSA; Serological Proteins Inc., Kankakee, IL, USA) in PBS (1% BSA-PBS) for 20 min to reduce non-specific staining. The sections were then incubated for 2 h at room temperature with: (1) rabbit antiserum against rat tissue nonspecific ALP, generated by Oda et al.14 (link) at a dilution of 1:100; (2) rabbit anti-PRX1 antibody (Abcam Ltd., Shanghai, China) at a dilution of 1:100; (3) rabbit anti-PRX5 antibody (Abcam Ltd., Shanghai, China) at a dilution of 1:100 in 1% BSA-PBS. After rinsing with PBS, the sections were incubated with horseradish peroxidase-conjugated swine anti-rabbit IgG (DaKo, Glostrup, Denmark) for 1 h at room temperature. The immunoreaction was visualized with diaminobenzidine (DAB) (Sigma-Aldrich, St. Louis, MO, USA). All sections were counter stained with methyl green and observed under a light microscope (Olympus BX53, Tokyo, Japan).

MC3T3-E1 cells were harvested at selected time points and lysed in RIPA lysis buffer (ComWin Biotech Co., Ltd., Beijing, China). Protein samples (50 μg) were mixed with 1/4 volume of 5 × SDS loading buffer and boiled at 95 °C for 5 min. Following sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), proteins were transferred to polyvinylidene difluoride (PVDF) membranes. Immunoblotting was performed with the following antibodies: rabbit anti-PRX1 antibody (Abcam Ltd., Shanghai, China) at a dilution of 1:1000, rabbit anti-caspase-3 antibody (Abcam Ltd., Shanghai, China) at a dilution of 1:2000 and mouse anti-GAPDH (Abcam Ltd., Shanghai, China) at a dilution of 1:2000, overnight at 4 °C. The secondary antibodies used were horseradish peroxidase-conjugated swine anti-rabbit IgG (DaKo, Glostrup, Denmark) for PRX1 and caspase-3, and horseradish peroxidase-conjugated rabbit anti-mouse IgG (Abcam Ltd., Hong Kong) for GAPDH, at room temperature for 1h. Western blot images were captured using a FluorChem E System (ProteinSimple, Santa Clara, CA, USA). The blots were cropped from different gels but all the gels had been run under the same experimental conditions.

MC3T3-E1 cells were harvested after corresponding treatment and lysed in RIPA lysis buffer (Beyotime, Beijing, China) containing protease inhibitors. Protein concentrations were determined using BCA protein assays (Beyotime, Beijing, China). A total of 30 μg of each sample were added and separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, then transferred to polyvinylidene fluoride membrane. The primary antibodies were added in 1:1000–5000 dilution and incubated at 4 °C overnight. Horseradish peroxidase-conjugated swine anti-rabbit IgG (DaKo, Glostrup, Denmark) was added and incubated at room temperature for 1 h. Western blot images were captured using a FluorChem E System (ProteinSimple, Santa Clara, CA, USA).