Hematoxyline

Paraffin embedded sections were deparaffinised in histoclear, hydrated in methanol and stained for transcription factors sex determining region Y-box 9 (SOX9) and collagen 2 (COL2). For SOX9, the specimens were incubated SOX9 antibody (1:200, Rabbit polyclonal, NovusBiologicals) with Biotinylated SP-conjugated goat anti-rabbit (1:500), Streptavidin Alexa 555 (1:500) (Jackson ImmunoResearch) and DAPI (1:2500, Sigma Aldrich). For COL2, the specimen were incubated with pepsin (1 mg/mL in 5mM HCl, Sigma Aldrich), 3% H2O2 (Chem-LAB), SOX9 antibody (1:200, MAB 8887, Millipore), goat anti mouse (1:500, Jackson ImmunoResearch), 3,3′-diaminobenzidine substrate (DAKO) and hematoxyline (SigmaAldrich). Specimen were mounted in Mowiol for confocal microscopic analysis using an Olympus FluoView FV1000 and visualized by the samples by Z-stacking 35 images of 26.22 micrometer. SOX9 nuclear translocation was artisanally semi-quantified by measuring whether DAPI positive nuclei were SOX9 positive using Image J software. For each condition 300 cells were counted.

All samples were fixed using 4% paraformaldehyde for 1 hour. To allow easy handling, the in vitro microaggregates for histological purposes were embedded in 2,5% agarose (Invitrogen). All samples were dehydrated, embedded in paraffin, cut into 5 micrometer section using a microtome (Microm HM360 Prosan) and stained using histology and immunohistochemistry unless otherwise stated. To visualize glycosaminoglycans samples were stained with acidic Alcian Blue (pH = 1, Merck) and counterstained with nuclear fast red (Vector Laboratories). General cell morphology was visualized using hematoxyline (SigmaAldrich) and eosin staining (Klinipath). To visualize general tissue morphology and highlight circulating erythrocytes sections were stained with Masson’s Trichrome (SigmaAldrich). Apoptotic cells were visualized using terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL) assay, which was combined with DAPI counterstaining to visualize all cells. Histological sections were microphotographed (X83P22F, Olympus). Semi-quantification was achieved with semi-automated analysis using ImageJ software.

Dulbecco’s modified Eagle’s medium (DMEM, high glucose), penicillin and streptomycin (PS) were purchased from Welgene (Dalseogu, Daegu, Korea), whereas fetal bovine serum (FBS) and horse serum are respectively from hyclone laboratories Inc (Logan, UT, USA) from Gibco Technologies Inc (Grand Island, NY, USA). Retinoic acid, linolenic acid (LA), dexamethasone, insulin, biotin, ascorbic acid, pantothenic acid, ascorbic acid, paraformaldehyde, hematoxyline, eosin, Triton X-100, 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) and fluoromount aqueous mounting media were obtained from Sigma Aldrich (St. Louis, MO, USA). Further, primary and secondary antibodies of both Donkey anti-goat immunoglobulin labelled with fluorescein isothiocyanate (IgG-FITC) and donkey anti-goat IgG-biotinylated were purchased from Santacruz biotechnology (Dallas, TX, USA) and primers from Macrogen.