Harris s hematoxylin

Paraffin sections of OSSN tissues samples were deparaffinized using serial solutions of xylene(catalogue no-35417, Thermo Fisher Scientific India Pvt Ltd, Mumbai, India)and hydrated through serial dilutions of 100%, 90%, 80% alcohol (catalogue no-26897, Thermo Fisher Scientific India Pvt Ltd, Mumbai, India). Then washed with running tap water and stained with Harris’s hematoxylin (catalogue no-H3136100G, Sigma aldrich, USA) for 10minutes, and washed with running tap water for 5minutes. They were further differentiated using 1% acid alcohol, washed with running tap water for 10 minutes, counter stained with1% Eosin Y (catalogue no-E4382, Sigma-aldrich, Germany) for 1 to 2 minutes, dehydrated with 80%, 90%, 100% alcohols and cleared with xylene, and mounted with DPX mount medium (catalogue no-46029 Fine-chem limited, Mumbai, India)[17 ].

Placenta and kidney tissue samples were fixed in a series of 10% formaldehyde, dehydrated graded alcohols, and embedded in paraffin. Placenta and kidney tissue sections (6 mm) were de-waxed, hydrated, and quenched with 0.3% H₂O₂ for 30 min. Kidney slices were stained with hematoxylin-eosin (HE) and periodic acid-Schiff (PAS). Then heat-induced antigen retrieval was accomplished, and the slides were incubated with PAS (a fibrinoid tissue marker), the anti-α-SMA antibody (1 μg/ml), and anti-cytokeratin antibody overnight at 4°C. After washing, the sections were incubated with HRP-labeled goat anti-rabbit immunoglobulin for 1 h at room temperature. Staining was completed by incubation with diaminobenzidine chromogen solution (DAB) (Santa Cruz; sc-2017). The sections were counterstained with Harris’s hematoxylin (Sigma-Aldrich, USA), dehydrated, and mounted. Corresponding concentrations of anti-IgG served as non-specific controls. Photographs were taken with a microscope (Leica Microsystems, Germany).

For periodic acid–Schiff (PAS) staining, the sections were deparaffinized and rehydrated. The slides were placed in 1% periodic acid (Sigma-Aldrich) for 20 min at room temperature (RT) and then in Schiff’s reagent (Sigma-Aldrich) for 20 min. After rinsing twice for 5 min under tap water, the slides were counterstained with Harris’s hematoxylin (Sigma-Aldrich). For the detection of DBA lectin, the histological sections were deparaffinized, rehydrated, and heated in 10 mM sodium citrate buffer (pH 6.0) by autoclave and were further incubated for 20 min with 3% hydrogen peroxide (Sigma-Aldrich) to quench the endogenous peroxidase activity. After washing with PBS, nonspecific binding was reduced by blocking the sections with 1.5% bovine serum albumin (BSA) in PBS for 1 h. The sections were incubated with biotinylated DBA lectin (Vector Laboratories, Burlingame, CA, USA) overnight at 4 °C. After washing, the slides were incubated with an avidin–biotin complex kit (Vectastain ABC kit; Vector Laboratories). The slides were incubated in fresh 3,3′-diaminobenzidine-HCl (DAB) and rinsed under tap water. The labeled tissue sections were then mounted and analyzed under a bright-field microscope (Nikon, Tokyo, Japan).

Chondrogenesis differentiation was assessed by micropellet formation. The differentiation tests were performed for 8 biological replicates, as indicated in Supplementary Table S1. In total, 5 × 10⁵ cells from passages were placed in a 15 ml conical tube and centrifuge at 1500 rpm for 5 min. The pellet was cultured in 2 ml of differentiating medium (StemProChondrogenesis Differentiation kit, Gibco by Life Technology) for six weeks, with medium changes twice a week. The pellet was fixed with 4% formaldehyde for 24 hr at RT, dehydrated by applying the ethanol in increasing concentration and embedded in paraffin. Five micron thick tissue histological sections were prepared using a microtome. The paraffin-embedded tissues were deparaffinized, hydrated and stained with 1% Alcian Blue pH 2.5 (Sigma-Aldrich, Saint Louis, Missouri, USA) in 3% acetic acid for 30 min at RT. After staining, the slides were counterstained with Harris’s hematoxylin (Sigma-Aldrich, Saint Louis, Missouri, USA) for 1 min at room temperature. Blue staining indicated synthesis of proteoglycans by chondrocytes^{55 (link),56} .

Cells were seeded into transwell filters (8  μM pores; BD Biosciences) and incubated for 24 h in media (as above except FCS was reduced to 1%) containing 0.1–10 μM S1P (Sigma-Aldrich, Poole, UK) or vehicle (methanol). Cells were methanol-fixed then those on the upper surface of the membrane were removed using a cotton swab and the remainder were stained with Harris's hematoxylin (Sigma). Cells were viewed by light microscopy (Olympus BX41 microscope,×10 magnification); the number of cells in 6 fields of view were counted. In some experiments, the S1PR-1/3 inhibitor, FTY720 or pFTY720 (100 nM, Cayman Chemical Company, Ann Arbor, USA), the S1PR2 inhibitor, JTE-013 (100 nM, Tocris, Abingdon, UK) or the Rho kinase inhibitor, Y27632 (10 μM; Cytoskeleton Inc., USA) was added to cultures for 30 min before treatment with 100 nM S1P for a further 24 h. In experiments to assess the affect of 1,25-dihydroxyvitamin D (D₃; Sigma) on EVT migration, D₃ (1-10 nM) was added for 48 h or 72 h before incubation with 100 nM S1P for a further 24 h.