Cultured NP cells were rinsed three times with PBS and fixed with 4% paraformaldehyde. After washing again with PBS, the cells were permeabilized with 0.5% Triton X-100 in PBS for 20 min and blocked with 5% FBS for 30 min. Thereafter, the NP cells were incubated overnight at 4 °C with antibodies against MMP-9 (1:100; Abcam) and then incubated with a Cy3-conjugated goat anti-rabbit IgG antibody (1:100; Boster Bio, Pleasanton, CA, USA) for 1 h at 37 °C. After washing in the dark, the cells were incubated with DAPI for 5 min. Cells were imaged using a fluorescence microscope (Olympus).
Antibodies against mmp 9
Manufactured by Abcam
Sourced in United States, United Kingdom
Antibodies against MMP-9 (Matrix Metalloproteinase-9) are laboratory reagents used to detect and quantify the presence of MMP-9 in various biological samples. MMP-9 is an enzyme involved in the breakdown of extracellular matrix and plays a role in various physiological and pathological processes. These antibodies can be used in techniques such as Western blotting, ELISA, and immunohistochemistry to study the expression and activity of MMP-9.
Automatically generated - may contain errors
Lab products found in correlation
Fluorescence microscope, by Olympus (1 mentions)
Cy3 conjugated goat anti rabbit igg antibody, by Boster Bio (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Tartrate resistant acid phosphatase staining, by Merck Group (1 mentions)
M csf, by R&D Systems (1 mentions)
Rankl, by R&D Systems (1 mentions)
3 protocols using antibodies against mmp 9
1
Immunofluorescent Localization of MMP-9 in Cultured NP Cells
2
Immunohistochemical Analysis of Protein Markers
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The tissue microarray blocks were used for immunohistochemistry staining. Briefly, the TMA blocks were immersed in 10 mmol/L of the EDTA buffer and reacted at 125°C for 10 minutes to retrieve the MMP‐9, p‐ERK1/2 and p‐P65 antigen. After 3% H2O2 in MeOH treatment and 3% BSA treatment, the slides were then incubated with antibodies against MMP‐9, p‐ERK1/2 and p‐P65 (Abcam) in cold temperature for a day. The IHC stains for the MMP‐9, p‐ERK1/2 and p‐P65 proteins were produced and a counterstain of haematoxylin was applied.
3
Murine Bone Marrow Macrophage Osteoclastogenesis
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The BMMs were obtained from the tibia and femur of mice following a previously described protocol [29 (link)]. BMMs were cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco, USA), 10% FBS, 100 U/ml penicillin (Gibco, USA) and M-CSF (30 ng/ml) (R&D, USA) for 12 h. The hydrogel sheets were placed in the upper chamber and the BMMs were collected in the lower chamber of transwell plates. RANKL (50 ng/ml) (R&D, USA) and M-CSF (30 ng/ml) were added to the culture media and cells were cultured for another 5 days. Tartrate-resistant acid phosphatase (TRAP) staining (Sigma USA) was performed on the 5th day. Antibodies against MMP-9 (Abcam Cambridge, UK) immunofluorescence staining was performed according to the aforementioned method. The supernatant was extracted for ELISA and the BMMs were collected in the lower chamber for RT-qPCR to detect the expression of osteoclast-related genes, including MMP-9, C-FOS, NFATc1. Actin was used as an internal reference, and the primer sequences were designed by Genewiz, as shown in Table 1 . Each experiment was repeated three times with three parallel samples (n = 3).
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