Hrp conjugated goat anti rabbit igg

The femur samples were first fixed for three days in a 4% formaldehyde buffer, then decalcified for 21 days in a 10% EDTA buffer. The samples were then sectioned into tissue slices with a thickness of 5 µm and immersed in paraffin. Hematoxylin and eosin (H&E) staining (Solarbio) was used following the established methodology to evaluate the histological morphology.
IHC staining of Bone γ-carboxyglutamate protein (Bglap) was carried out for in vivo quantification of osteoblastogenesis The sections were dewaxed, hydrated, and subjected to antigen retrieval by microwave heating method at 96 °C for 30 min. The sections were then subjected to treatment with 0.1% Triton X-100 for 30 min, followed by blocking with 5% BSA for another 30 min at room temperature after cooling. The samples were treated overnight at 4 °C with rabbit-derived anti- Bglap (1:200; Bioss, China). HRP-conjugated goat anti-rabbit IgG (1:500; Bioss, Beijing, China) was employed as secondary antibodies the next day. Positive signals were detected through the utilization of the universal DAB color development kit (Beyotime, Jiangsu, China). The integral optical density (IOD) of Bglap positive signals across all sections was determined by Image-Pro Plus software (Media Cybernetics, Rockville, MD, USA). All the results were obtained from a minimum of six randomly selected fields for each sample.

Western blot was performed as previously described with minor modifications (Zhang et al., 2019 (link)). Briefly, harvested cells were lysed in RIPA buffer, and protein concentrations were determined via BCA assay follow the manufacturer's instructions. Total protein (50 μg) was resolved (SDS‐PAGE, 8%–15% gradient gels) and transferred to PVDF membranes. Immunoblotting was carried out using primary antibodies and horseradish peroxidase‐conjugated secondary antibodies. The blots were visualized using a Tanon 5200 automatic chemiluminescence image analysis system (Tanon, Shanghai, China).
The following antibodies were used: anti‐ERK (1:1000, cat no. 4695S, CST), anti‐p‐ERK (1:500, cat no. 9101S, CST), anti‐PI3K (1:1000, cat no. 4249S, CST), anti‐p‐PI3K (1:500, cat no. 4228S, CST), anti‐GAPDH (1:2000, cat no. UM4002, UtiBody), HRP‐conjugated goat anti‐rabbit IgG (1:3000, cat no. bs‐0295G‐HRP, Bioss) and HRP‐conjugated goat anti‐mouse IgG (1:3000, cat no. bs‐0296G‐HRP, Bioss).

Total protein from chicken brain tissues was extracted using SDS Lysis Buffer (Beyotime, China). The concentrations of the protein extracts were measured and calculated using the Enhanced BCA Protein Assay Kit (Beyotime, China). Equal amounts of protein from each extract were subjected to 12% SDS-PAGE gel electrophoresis. Separated proteins were transferred to nitrocellulose (NC) membranes in Tris-glycine buffer for 1 h at 100 mA. The NC membranes were blocked with 5% skim milk at 37 °C and 50 rpm for 4 h and incubated overnight with the diluted iNOS primary antibody (1:1000, provided by Dr. Xu) and GAPDH antibody (1:1000, Beyotime, China) followed by a 1 h incubation with a horse-radish peroxidase (HRP)-conjugated goat anti-rabbit IgG (1:5000, Bioss, Beijing) at 37 °C and 50 rpm. The signals were detected by X-ray film (Kodak, USA), and the corresponding protein expression levels were calculated according to greyscale values of the iNOS and GADPH bands.