The meniscus explants were fixed in Z-Fix (Anatech). Paraffin-embedded sections (5–7 µm) were stained with Safranin-O to detect glycosaminoglycan. Fast green was used as a counterstain. the following antibodies were used: MKX (HPA006927, 1:200, Sigma-Aldrich), COL1A1 (AB292, 1:200, Abcam), COL2A1 (II-II6B3-S, 1:50, DSHB), ACAN (L0101, 1:50, Assay Biotechnology), MMP13 (AB39012, 1:600, Abcam), COL10A1 (X-AC9, 1:10, DSHB) and RUNX2 (SC-390351, 1:75, Santa Cruz). Mouse IgG (I-2000, Vector Laboratories, Inc.) or Rabbit IgG (I-1000, Vector Laboratories, Inc.) were used as negative controls (fig. S16 ). There was only weak staining for COL1A1 and COL2A1 in DMS compared with bovine meniscus explants (fig. S17 ). For quantitative analysis of DAPI (H-1500 Vector Laboratories, Inc.) staining, the cells in the DMS and at the borderline between DMS in injured explants were counted. Polarized picrosirius red staining and DIC microscopy were used to detect collagen types and fiber alignment within the explants and to examine the fibrous interconnectivity between inserted DMS and injured explants. Histopathological scoring of meniscus, articular cartilage, and synovium was analyzed by previous methods (51 (link)–54 (link)).
Protocol detail
Histological Characterization of Meniscus Explants
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Antibodies
Articular cartilage
Assay
Bovine
Collagen
Dapi
Fast green
Fibrous
Glycosaminoglycan
Meniscus
Microscopy
Mmp13
Mouse
Paraffin
Rabbit
Runx2
Safranin o
Synovium
Vector
Weak
Corresponding Organization : Scripps Institution of Oceanography
Other organizations : Scripps Clinic
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Variable analysis
independent variables
- Fixation of meniscus explants in Z-Fix
- Paraffin-embedding and sectioning (5-7 µm) of explants
- Staining with Safranin-O and Fast green
- Immunohistochemical staining for MKX, COL1A1, COL2A1, ACAN, MMP13, COL10A1, and RUNX2
- Glycosaminoglycan content (Safranin-O staining)
- Expression of meniscus-related markers (MKX, COL1A1, COL2A1, ACAN, MMP13, COL10A1, RUNX2)
- Cell count in DMS and at the borderline between DMS and injured explants (DAPI staining)
- Collagen types and fiber alignment (Polarized picrosirius red staining and DIC microscopy)
- Histopathological scoring of meniscus, articular cartilage, and synovium
- Paraffin-embedded sections (5-7 µm) of meniscus explants
- Negative controls using mouse IgG or rabbit IgG
- Bovine meniscus explants (for COL1A1 and COL2A1 expression)
- Mouse IgG (I-2000, Vector Laboratories, Inc.)
- Rabbit IgG (I-1000, Vector Laboratories, Inc.)
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