Chondroitinase abc

Cryosections were washed for 10 min with dH₂O to remove the cryocompound, transferred to absolute methanol for 20 min and washed twice in 0.01% Tween 20 in PBS (PBS-T). Enzymatic treatment by 1U/ml Hyaluronidase (Sigma-Aldrich, H3506) and 0.25 U/ml Chondroitinase ABC (Sigma-Aldrich, C2905) in PBS-T for 30 min at 37 °C allowed the digestion of matrix. After washing in PBS-T, pellets were transferred in blocking solution containing 5% horse serum (Vector laboratories, S-2000) in PBS-T for 30 min at room temperature. Primary antibody anti-type II collagen (4 μg/mL, CIICI, see acknowledgement section) and anti-type I collagen (1:400, Origene Acris, R1038) were added over night at 4 °C. Slides were washed with PBS-T, then the secondary antibodies were added, 4 μg/ml anti Rabbit Alexa Fluor 488 against type I collagen (Thermo Fisher, A-11008) and 5 μg/ml anti Mouse Alexa Fluor 660 against type II collagen (Thermo Fisher, A-21055) for 1 h at 37 °C. After washing with PBS-T, the nuclei were counterstained with 2-(4-Amidinophenyl)-1H-indole-6-carboxamidine (DAPI) 2.5 μg/mL and then cover slipped with ProLong mounting solution (Thermo Fisher, P10144).

CEMIP localization was assessed by immunohistochemistry in knee joint sections using anti-KIAA1199 antibody (1:1000) as previously described [10 (link)]. Deparaffinized sections were pretreated with chondroitinase ABC (1 U/mL; Sigma-Aldrich) at 37 °C for 2 h. The endogenous peroxidase was blocked with 3% H₂O₂ in PBS at 20 °C for 15 min and incubated in normal goat serum at 20 °C for 60 min. Rabbit anti-aggrecan neo antibody (10 mg/mL) was used as the primary antibody at 4 °C for 16 h. Histofine simple stain rat MAX PO(R) (Nichirei, Tokyo, Japan) was used as the secondary antibody [40 (link)]. The reaction was visualized by diaminobenzidine (DAB; Histofine simple stain DAB, Nichirei), resulting in a brown color. Counterstaining was carried out with hematoxylin. Sections incubated with normal rabbit non-immune serum or incubated without primary antibody were used as negative controls.

Tissue sections (7 μm) from formalin-fixed, paraffin-embedded ankle joints of IL-1Ra^–/– mice and BALB/c control mice were digested with proteinase-free chondroitinase ABC (0.25 units/ml in 0.1 M Tris-HCl, pH 8.0; Sigma-Aldrich) for antigen retrieval. Tissue sections were incubated overnight with rabbit anti-S100A8 and anti-S100A9 [23 (link)] or with rabbit anti-VDIPEN for staining of MMP-mediated cartilage destruction. Sections were then incubated with biotinylated horseradish peroxidase-conjugated goat anti-rabbit IgG (Dako) as a second antibody followed by incubation with avidin-streptavidin-peroxidase (Elite-kit, Vector). Peroxidase activity was assessed by staining with 3,3′-diaminobenzidine (DAB; Powervision DAB, Immunologic, Duiven, the Netherlands) in the presence of H₂O₂ and all sections were counterstained with hematoxylin for S100A8 and S100A9 staining and with orange G (2 %) for VDIPEN staining.

In accordance with the predominant metastatic pattern of ovarian carcinomas in the abdomen, we studied SKOV3 cell behavior not only as single cells but also as tumour cell aggregates (spheroids). Multicellular spheroids are thought to represent an important way for tumour dissemination in ovarian cancer. In addition, compact spheroid formation provides a protective shield for tumour cells. For that purpose, cells from subconfluent cultures were detached with 2 mM EDTA and multicellular spheroids were generated using the hanging-drop method [34] (link). In brief, cells were resuspended in DMEM growth medium supplemented with 10% methylcellulose to a final cell concentration of ±4×10⁴ cells/ml and incubated in hanging droplets (25 µl) overnight. After cell aggregation, spheroids were washed in phosphate buffered saline (PBS) and used for migration assays. To further study the adhesive properties of CS wildtype SKOV3 cells in hanging drops were digested with specific glycosaminoglycan degrading enzymes including chondroitinase ABC (Sigma Aldrich, St. Louis, USA), chondroitinase B and chondroitinase AC (IBEX Technologies, Montreal, Canada).

The following materials were purchased from the manufacturers indicated: heparin, heparan sulfate, chondroitin sulfate A, B and C, heparinases I and III, chondroitinase ABC, fluorescein isothiocyanate (FITC), GenElute PCR clean-up kit, phospholipase C phosphatidylinositol-specific (PI-PLC) from Bacillus cereus, all from Sigma-Aldrich (St. Louis, MO, USA); 2-O, 6-O and N-desulfated heparins from Amsbio (Abingdon, UK); Dulbecco’s Modified Eagle’s minimal essential medium (DMEM) and Minimum Essential Medium (MEM), fetal bovine serum, penicillin-streptomycin, and PBS-phosphate-buffered saline from Gibco-Thermo Fischer Scientific (Waltham, MA, USA); Brain-Heart Infusion broth from Pronadisa (Madrid, Spain); RNeasy Kit and RNase-Free DNase from Qiagen (Hilden, Germany); High-Capacity cDNA Reverse Transcription Kit and PowerSYBR Green PCR Master Mix from Applied Biosystems (Foster City, CA, USA). Synthetic peptides were from Abyntek Biopharma (Derio, Spain); mouse monoclonal anti-syndecan 1 (CD138) from DakoCytomation (Carpinteria, CA, USA); and rabbit anti-syndecan 2, goat anti-syndecan 3 and rabbit anti-syndecan 4 polyclonal antibodies from Santa Cruz Biotechnology (Santa Cruz, CA, USA). All other chemicals were obtained from commercial sources and were of analytical grade.

Adult male Wistar rats were injected with either SKF38393 (5 mg/kg body weight, i.p.) or vehicle as described previously [36 (link)]. Rats were anesthetized with isoflurane 1 h after injection, followed by decapitation with a guillotine. For further use, the prefrontal cortex (PFC), hippocampus and rest of the brain were dissected and stored at −80 °C, as described in detail elsewhere [37 (link)]. Subcellular brain fractionation was performed according to Reference [38 (link)]. Synaptosomal fractions were harvested and incubated with Chondroitinase ABC (Sigma-Aldrich, St. Louis, MO, USA) at 37 °C for 30 min.