After imaging, the animals were sacrificed and the each knee was dissected aseptically, and fixed in 4 % paraformalin, then decalcified in EDTA, which was changed every 5 days. The decalcified knee was cut in the mid-sagital plane, and paraffin-embedded. Serial knee sections of exact 5 μm thickness from the middle part of the knee were obtained to prepare slides. Rat knee joints were stained with hematoxylin and eosin (H&E) to assess general morphology and neovascularization. For immuno-histochemistry, Anti-CollagenIIprimary antibody (Abcam) was used in 1:500 dilution. All samples from both knees were stained, and examined independently by two observers. Sections were stained for GAGs with 0.5 % Safranin-O and 0.2 % Fast Green counterstain. For each sample, three sagittal sections were used for thickness histomorphometry analysis and digital images of each section were captured.
Anti collageniiprimary antibody
Manufactured by Abcam
The Anti-Collagen II primary antibody is a tool used in research applications to detect and measure the presence of collagen type II, a major structural component of cartilage and other connective tissues. This antibody can be used in various immunoassay techniques to identify and quantify collagen II in biological samples.
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2 protocols using anti collageniiprimary antibody
1
Histological Analysis of Rat Knee Joints
2
Chondrogenic Differentiation of Rabbit Chondrocytes
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To induce chondrogenic differentiation, 3×10 5 rbHP-PCs in a 15 ml polypropylene conical tube (Thermo Fisher Scientific) were seeded and a 160 g centrifuge for 7 minutes was performed. The cells were cultured in a chondrogenic differentiation medium (Cambrex Biosciences Inc.) composed of 10% FBS, 10 ng/ml TGF-β3, 100 nM dexamethasone, 25 μg/ml ascorbate, and 100 μg/ml sodium pyruvate. 18 In 24 to 48 hours, the cells gathered to form a 3D aggregate shape. Culture medium was changed every 3 days for 21 days.
The chondrocytes that differentiated into a 3D aggregate were fixed in 4% paraformaldehyde and embedded in paraffin. After sectioning into 4 um, staining was done using hematoxylin and eosin. The slides were stained with Alcian blue 8GX solution (Sigma-Aldrich) for 30 minutes and then washed two or three more times with distilled water and then stained further with hematoxylin.
The slides were stained with anti-collagen II primary antibody (1:200, Abcam) overnight at 4°C. The secondary antibody (1:1000, Abcam) was incubated for 30 minutes at room temperature. Finally, the slides were stained using 3,3-diaminobenzidine solution (Dako, Ft. Collins, CO).
The chondrocytes that differentiated into a 3D aggregate were fixed in 4% paraformaldehyde and embedded in paraffin. After sectioning into 4 um, staining was done using hematoxylin and eosin. The slides were stained with Alcian blue 8GX solution (Sigma-Aldrich) for 30 minutes and then washed two or three more times with distilled water and then stained further with hematoxylin.
The slides were stained with anti-collagen II primary antibody (1:200, Abcam) overnight at 4°C. The secondary antibody (1:1000, Abcam) was incubated for 30 minutes at room temperature. Finally, the slides were stained using 3,3-diaminobenzidine solution (Dako, Ft. Collins, CO).
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