At the time of killing, the knee joints of mice were fixed in 4% paraformaldehyde, decalcified with 12.5% EDTA (pH 7.0) and embedded in paraffin. The knee joint sections (7 μm) were stained with H&E and safranin O and fast green. For the immunostaining, the sections were incubated with primary antibodies to mouse nestin (1:800; Abcam, Cambridge, MA, USA), osterix (1:1000; Abcam), LRG1 (1:100; Abcam), CD31 (1:100; Abcam), TNF-α (1:100; Abcam) and MMP13 (1:200; Bioworld Technology) overnight at 4 °C. These samples were incubated with goat antirabbit secondary antibodies conjugated with horseradish peroxidase (HRP).
Mouse nestin
Manufactured by Abcam
Sourced in United States
Mouse nestin is a protein expressed in neural stem cells and is commonly used as a marker for these cells. It plays a role in the maintenance and differentiation of neural stem cells.
Automatically generated - may contain errors
2 protocols using mouse nestin
1
Histological Evaluation of Mouse Knees
2
Immunostaining of Differentiated Cell Monolayers
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Monolayer cultures of differentiated cells were rinsed with phosphate-buffered saline (PBS) and incubated for 10 min at room temperature in PBS (pH7.4) containing 4% paraformaldehyde. After each of three rinses with PBS and 5% normal donkey serum (Abcam, Cambridge, MA, USA) in PBS, the cells were blocked for 2 h at room temperature with PBS containing 5% donkey serum and 0.3% Triton X-100. The cells were then incubated with mouse NESTIN (1:200), mouse C/EBPβ (1:100) (Abcam, USA) primary antibodies in blocking solution at 4°C overnight. After each of three rinses with PBS and blocking solution, the cells were incubated in blocking solution for 30 min at room temperature. After incubation of the cells in blocking solution with donkey anti-mouse IgG Cy3 conjugated secondary antibody (1:500) (Millipore, USA) for 2 h at room temperature in the dark, the cells were washed with PBS. Finally, the cells were counterstained with 4′,6-diamidino-2-phenylindole (DAPI)(1 μg/mL) (Invitrogen, Waltham, MA, USA), and images were acquired using a LSM 510 META Confocal Laser Scanning Microscope.
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