Fig 1: Examples of immunostained formalin fixed paraffin embedded intervertebral disc tissue sections. A, Aquaporin 7 (Abcam ab85907) cell membrane staining, canine nucleus pulposus (NP) tissue. B, Cytoplasmic Indian Hedgehog (Santa Cruz sc-1196) staining, human NP tissue. C, Nuclear localisation of TonEBP (Abcam ab3446), human NP tissue. D, Pericellular and matrix staining of collagen type II (Abcam ab34712), human NP tissue. E, Staining of aquaporin 6 (Abcam ab191061) indicating cartilaginous endplate (CEP) localisation, canine IVD tissue. F, CD31 (Abcam ab28364) staining within human annulus fibrosus (AF) tissue indicating the presence of endothelial cell infiltration. G, CCL3 (Abcam ab32609) staining within cell clusters, human NP tissue. H, Mouse IgG3, Kappa isotype control (Abcam ab18394), human NP tissue. I, Rabbit IgG isotype control (Abcam ab37415), human NP tissue. A, B, E, F, G, H, I scale bar = 100 µm. C, D scale bar = 50 µm
Fig 2: Differentiation of ASCs. (a, b) Adipogenic differentiation of ASCs, Oil Red stain, (15 days of cultivation; objective lens 20×, eyepiece 10×; phase contrast). (a) control — culture of third passage ASCs without using a differentiating medium, the subconfluent monolayer is formed by typical fibroblast-like cells. In the cells of the control culture, the formation of fat vacuoles is not fixed. (b) the culture of ASCs after cultivation in a differentiating medium. Fat culture vacuoles are clearly defined in culture cells stained with a specific Red Oil dye. The presence of fat vacuoles in the cells indicates the ability of ASC to differentiate in the adipogenic direction; (c, d) Osteogenic differentiation of ASCs. Osteogenic differentiation of ASCs, stained with alizarin red (15 days of cultivation; objective lens 10×, eyepiece 10×, light microscopy). (c) The control culture of ASCs is presented as a subconfluent monolayer formed by morphologically identical spindle-shaped cells, with no calcium deposits. (d) The culture of ASCs after cultivation in a special differentiating medium. The culture is a subconfluent monolayer. Calcium deposits are clearly visualized, which are stained with a differentiating dye, alizarin red. Calcium deposits indicate the ability of ASC to differentiate in the osteogenic direction; (f) Chondrogenic differentiation of ASCs. Staining of type II collagen deposits in the pellet with polyclonal antibodies (Abcam, ab34712; cell nuclei stained with hematoxylin and eosin (Sigma-Aldrich, Germany); objective lens 20×, eyepiece 10×). Ball formation and deposition of type II collagen in them indicates chondrogenic differentiation of ASCs.
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