Trap alp stain kit

Mouse femurs were fixed in 10% buffered neutral formalin (Sigma, MO, USA) for 24 h followed by decalcification in 10% EDTA (0.5 mol·L^–1) at 4 °C until complete decalcification was confirmed by x-ray analysis. Hematoxylin–Eosin (H&E) staining was performed on 6 μm sections of decalcified paraffin embedded femurs to assess bone marrow histology. For osteoclast detection in femurs, Tartrate-resistant acid phosphatase (TRAP) staining was performed using TRAP/ALP Stain Kit according to the manufacturer’s instructions (#294-67001, Wako Chemicals, USA).

For histomorphometry, nonfixed and undecalcified left femurs were used for frozen section by the Kawamoto’s film method. The frozen blocks were cut into 5 µm slices by a Leica CM3050 S cryostat (Leica Biosystems, Wetzlar, Germany). Hematoxylin and eosin (HE) staining was performed in accordance with standard protocols. We measured cortical bone width at four randomly selected locations on HE of the overall femur by using analysis application hybrid cell count (Keyence, Osaka, Japan). Tartrate-resistant acid phosphatase (TRAP) was stained by TRAP/ALP Stain Kit (FUJIFILM WAKO, Osaka, Japan). Purified anti-mouse panendothelial cell antigen antibody (MECA-32, BioLegend, USA) was used for the detection of vascular endothelial cells. Microscopic images were captured in tiled array by a semi-automated BZ-X710 microscope (Keyence, Osaka, Japan) and analyzed by NIH ImageJ. The regions for the analysis of bone resorption and formation in histomorphometry were measured by photographing interest three section (x100) of the femoral diaphysis in each mouse. The following parameters were measured: number of osteoblasts per bone surface (Ob.N/BS), eroded surface per bone surface (ES/BS), number of osteoclasts per bone surface (Oc.N/BS), and surface of osteoclasts per bone surface (Oc.S/BS).

Bone marrow cells were isolated from 8- to 10-week-old control and Elovl6^-/- mice. Femurs and tibias were removed and dissected free of adherent soft tissues. The bone ends were cut, and the marrow cavity was flushed out with DMEM from one end of the bone using a sterile 21-gauge needle. The bone marrow was carefully dispersed by pipetting and incubated overnight in DMEM containing 10% FBS for 20h, and non-adherent cells were harvested and inoculated at 1×10⁵ cells/cm² for 2 days in the presence of 10 ng/ml of M-CSF (R&D systems, Minneapolis, MN). Adherent cells were used as bone marrow macrophages (BMMs). To obtain osteoclastic cells, BMMs were further cultured with 100 ng/ml of RANKL (R&D systems) in the presence of 50 ng/ml of M-CSF for 5 days and cells were then stained with TRAP using TRAP/ALP stain kit (Wako Pure Chemicals, Osaka, Japan) [17 (link)]. TRAP-positive multinucleated cells with more than 3 nuclei were counted as osteoclasts.

Tilapia bone was provided from Ocean King Fisheries Co. Ltd. (Yunnan, China). Papain, pepsin, flavoring protease, neutral protease, complex protease, basic protease, and trypsin protease were provided by Shanghai Yuanye Biological Technology Co. Ltd. (Shanghai, China), and animal protease was provided by Pangbo Biological Engineering Co. Ltd. (Nanjing, China). Peptides were produced by Shanghai Synpeptide Co. Ltd. (Shanghai, China). Hydrolytic protease was obtained from Novozymes Biotechnology Co. Ltd. (Beijing, China). Acetonitrile and trifluoroacetic acid (TFA) of mass spectroscopic grade were obtained from Merck KGaA (Darmstadt, Germany). Raw 264.7 and MC3T3-E1 cells were obtained from the Kunming Institute of Zoology cell bank. Tartrate resistant acid phosphatase (TRAP) kit, acid phosphatase (ALP) kit, alizarin red staining kit, and BCA Protein Assay Kit were obtained from Beyotieme Co. Ltd. (Shanghai, China). TRAP/ALP stain kit was obtained from Wako Co. Ltd. (Wako, Japan). All the other chemicals and reagents were of analytical grade.

Joint tissues were decalcified in 10% EDTA in 0.1 M Tris buffer (pH 7.4), fixed in 4% paraformaldehyde, and embedded in paraffin. Tissue sections were stained with hematoxylin/eosin, and also stained for TRAP using the TRAP/ALP stain kit (Wako Pure Chemical Industries Ltd.).