Alginate culture

Chondrocytes were encapsulated in alginate beads immediately after isolation according to the method of Häuselmann et al. (Häuselmann et al., 1994). Briefly, cells were suspended in 1,2% sodium alginate (Sigma-Aldrich Co., Taufkirchen, Germany) in 150 mM NaCl (4x10⁶ cells/ml alginate solution). The chondrocyte suspension was passed drop-wise through a 22-gauge needle into a 102mM CaCl2 solution under constant stirring. Following 10 min polymerization, beads were washed with 150 mM NaCl and with DMEM/F12.

Chondrocytes in alginate beads were cultured in the same medium and culture conditions as cartilage pieces and cells in monolayer.

When required, alginate beads were dissolved by exposure to solubilization buffer (55 mM sodium citrate and 150 mM NaCl, pH 6,00) at 37°C for 10 -15 min. The cells were recovered by centrifugation.

3.3. human Mesenchymal Stem Cells (hMSCs) from bone marrow

Frozen hMSCs from healthy donors (age 18-30 y.) were purchased from Cambrex (Cambrex Bio Science, Verviers, Belgium). Cells were plated in 75 or 150 cm² flasks and cultured in Mesenchymal Stem Cells Growth Medium (MSCGM), a low glucose Dulbecco's modified Eagle's medium supplemented with L-glutamine, penicillin, streptomycin and fetal calf serum (all from Cambrex). Cells were fed every 3-4 days.

When the cells reached 80% confluency they were passaged.

The cells used for chondrogenic differentiation procedure were derived from the 5^th passage.

3.3.1. Chondrogenic differentiation

For chondrogenic differentiation 25x10⁴ hMSCs were placed in a 15-ml conical polypropylene tube, washed with Incomplete Chondrogenesis Induction Medium (ICIM) consisting of Differentiation Basal Medium Chondrogenic (Cambrex) supplemented with 1 mM sodium pyruvate, 0.17 mM ascorbic acid–sodium salt, proline, glutamine, 100nM dexamethasone , 1% ITS+Premix, and antibiotics: Pen/Strep (supplementation available from Cambrex as Chondrogenic SingleQuots). After washing with ICIM cells were resuspended in 0.5-ml complete chondrogenic medium (CCM) consisting of supplemented ICIM and 0,01 µg/ml TGF-β3 (R&D Systems, Wiesbaden, Germany).

Cells were centrifuged at 1000 rpm for 5 minutes at RT. The pellets were maintained in culture with 1 pellet/tube and 0.5 ml CCM/tube. Medium was changed every 2–3 days.

3.3.2. Osteogenic differentiation

For osteogeneic differentiation, hMSCs were seeded in 6-well plates: 3x10⁴ cells in 2ml MSCGM per well. After 6 hours when cells were adherent MSCBM was replaced by Osteogenic Induction Medium, containing 100nM dexamentasone, 0,05mM ascorbate and 10 mM ß-glycerophosphate (all from Cambrex). Control wells were cultured in MSCBM to the end of the experiment.

3.3.2.1. Assessment of osteogenic phenotype

3.3.2.1.1. von Kossa staining

Cells were fixed with 60% acetone buffered with citrate for 30 sec followed by washing twice in deionized water. The fixed cells were incubated with 2% silver nitrate under direct light from a 60-W lamp for 1h and afterwords with 2,5% sodium thiosulfate for 5 min, rinsed with deionized water and counterstained with 0,33% neutral red for 5 min, finally rinsed in tap water and mounted with Aquatex (Merck) prior to examination under the microscope.

3.3.2.1.2. Alkaline phosphatase staining

Cells in wells of the 6-well plates were fixed with citrate buffered 60% acetone for 30 sec, then gently rinsed in deionized water for 45 sec and stained with a mixture of Fast Violet B salt and Naphthol AS-MX Phosphate Alkaline Solution for 30 min, according to the manufacturer’s instructions (Sigma, Procedure No. 85). Afterwards the cells were counterstained with Mayer’s hematoxylin (Sigma) for 10 min, rinsed in tap water and mounted with Aquatex prior to examination under the microscope.

3.3.3. Adipogenic differentiation

hMSCs were plated at 200,000 cells/well in a 6-well plate and grown to confluence in MSC medium. Subjecting confluent monolayers to 3 rounds of adipogenic treatment induced adipogenic differentiation. Each round consisted of 48–72 hours in adipogenic induction medium: DMEM-high glucose (4,5g/l), 1µM dexamethasone, 0,2mM indomethacin, 0,5 mM 3-isobutyl-1-methyl-xanthine, 0,01 mg/ml insulin and 10% FCS, followed by 48–72 hours in maintenance medium: DMEM-high glucose, 10% FCS and 0,01 mg/ml insulin. Cells were assayed after an additional week in maintenance medium. Control wells were cultured in MSCBM to the end of the experiment.

3.3.3.1. Assessment of adipogenic phenotype 3.3.3.1.1. Oil red “O” staining

Cells were fixed with 4% formaldehyde in PBS for 10 min followed by washing twice with PBS. The cells were pretreated with 60% 2-propanol for 2min and then stained with 0,2% oil red “O” in 60% 2-propanol for 10 min. The cells were washed with 2-propanol and then with PBS. Finally, to stain nuclei, the cells were treated with Mayer`s hematoxylin for 10 min and washed in tap water. Coverslips were mounted using AquaTex.

3.4. Cell treatment with growth factors, cytokines and inhibitors

For experiments human recombinant cytokines and growth factors were used (all purchased from R&D Systems).

TGF-β3 used for chondrogenic differentiation was dissolved in 10mM HCl / 10%

ethanol to prepare 20µg/ml stock. Siliconized tips and tubes were used to prevent adhesion of TGF molecules to plastic. TGF-β3 was stored after reconstitution at -80°C for not longer as one month.

Cytokines and LPS (Sigma) were dissolved in PBS and stored at –20°C.

Cytokine Mix “promoter” consisted of:

TNFα 500 ng/ml IFN γ 10 000 U/ml IL-1ß 50 nM NO donors:

Stocks of NO donors were freshly prepared prior to cell stimulation.

Spermine-NONOate and DETA NONOate (Cayman) were dissolved in 10mM NaOH pH12,5 and SIN-1 (Sigma) was dissolved in PBS.

Arachidonic acid (AA) was purchased from Cayman. AA was supplied as a solution in ethanol. To change the solvent ethanol was evaporated under a gentle stream of nitrogen and dissolved in DMSO. Stock solution was stored at –20°C.

Inhibitors:

Inhibitors were dissolved in appropriate solvent. In most cases stock of inhibitor was prepared in DMSO and diluted to required concentration in assay medium. Final concentration of DMSO did not exceeded 0,1%. To exclude any solvent influence on the experiment control cells were treated with vehicle.

The cells were incubated with inhibitors 30 minutes before stimulation with cytokines.

3.4.1. Stimulation of chondrocytes

Chondrocytes in monolayer were seeded in 6-well or 24 well plates chondrocytes in alginate beads and chondrogenic pellets were placed in 24-well dishes.

Cells (calculated 1 bead/100µl medium) were stimulated with human 1nM IL-1β for 24h;

when necessary cells were preincubated with substances for 30 min and then stimulated for next 24h with IL-1β.

3.4.2. Stimulation of hMSCs

At indicated differentiation days hMSCs were stimulated with 1nM IL-1β for 24h (6 pellets/well, 0,75 ml medium/well: Bio Whittaker BE12-917, with 4,5 g/l Glucose, w/o L-Glutamine and Phenol Red + HMSC Chondrogenic SingleQuots).

After 24h of incubation, Griess assay was performed and the rest of supernatant was frozen for prostaglandins assessment. Pellets were harvested and resuspended in RNA-isolation buffer (RLT Buffer, Qiagen), than frozen at -80°C.

3.5. Biochemical methods

3.5.1. Determination of cellular viability

Viable cells werecounted in a Neubauer chamber by the trypan blue exclusion method.

3.5.2. Toxicity: release of lactate dehydrogenase (LDH)

The toxicity was determined by measuring the LDH activityusing a CytoTox 96 Non-Radioactive Cytotoxicity Assay (Promega, Mannheim, Germany).

50 µl of cell cuture media were used as samples, the assay was performed according to the manufacture's instructions. As positive control cells were incubated with 1% triton X-100 and the result was calculated as X-100% of LDH release (total).

3.5.3. Griess assay

150µl of cell culture supernatant was mixed with 10 µl 1% sulphanilamide (Sigma) in 0.1N HCl and 10 µl 0.1% N-(1naphthyl)-ethylenediamine (Sigma). Absorbance was read at 544 nm in reference to 690 nm in a microplate reader. Nitrite (NO2-)

concentrations were calculated by using a NaNO2 standard curve (0-35 µM) in cell culture medium.

3.5.4. PGE2 ELISA

Chondrocyte culture media were collected and frozen at –20°C.

The amount of PGE2 was determined with a commercially available ELISA kit (R&D Systems), according to the manufacture's instructions. Triplicate assays per sample were performed, and the data were converted to μg/ml.

3.5.5. GC/MS/MS

These measurements were performed in Marburg by Dr. Horst Schweer.

In parallel concentrations of COX-products in cell culture supernatants were determined using specific gas chromatography/triple stage quadrupole mass spectrometry (GC/MS/MS) as described by Schweer et al. (Schweer et al., 1994).

3.5.6. Cytochrome c assay

Superoxide production was measured using the cytochrome c reduction assay. 75µM cytochrome c was added to the cell culture media of chondrocytes, after incubation time absorption measurements were made in cell culture media using wavelength 550nm (Spectramax 384plus, Molecular Devices), the absorption maximum of reduced cytochrome c.

3.5.7. Electron Spin Resonance (ESR)

For measurement of superoxide production chondrocytes grown in monolayer were washed with PBS, scraped from the plastic surface and resuspended in ESR buffer.

Equal cell numbers were used for eachexperiment.

The high cell permeable and non-toxic spin trap: 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine hydrochloride (CM-H), was freshly prepared under N2 and added to a final concentrationof 1mM.

Table 5. The composition of ESR buffer (100ml).

Substance: Amount: Final concentration:

NaH2PO4 235mg 8,75mM

Na2HPO4 761mg 63mM

Glucose 100mg 0,1%

NaCl 15mg 2,5mM

KCl 37mg 5mM

CaCl2 20mg 1,8mM

Samples without cells but with spin trap and with polyethylene-glycolated superoxide dismutase (PEG-SOD) 50U/ml 100U/ml were measured as internal control. Spectra were recorded at room temperature in a 15µl glass-capillary (Brand) usinga Bruker e-spin spectrometer (Bruker, Rheinstetten, Germany)with the following parameters:

Field

Center field: 3487 G Sweep width: 65G

Resolution: 512 points Microwawe

Frequency: 9.776 GHz Power: 3.964 mW Receiver :

Receiver gain: 2.24e+002 Phase: 0.36 deg Harmonic: 1

Mod. frequency: 86 kHz Mod. amplitude: 2.06G Signal channel:

Conversion. 10.240 ms Time constant: 10.240 ms Sweep time: 5.243 s

3.5.8. SDS-Page

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)was performed as described in the work of Laemmli (Laemmli, 1970). Composition of gels is given in a table below (Table 6). Samples were boiled for 3 min at 95°C in Laemmli buffer and shortly centrifugated. Finally, samples and protein standards were loaded onto SDS-PAGE gels. As a molecular weight marker See Blue Plus2 (Invitrogen) was used. Gels were run at 50-170 V in the standard electrophoresis cell (Biometra) or Novex Mini-Cell (Invitrogen).

Table 6. Recipes for SDS-PAGE gel mixtures.

Stacking gel 5%

Separating gel 8%

Separating gel 10%

H2O 1,4 ml 3,6 ml 3,1 ml

Tris/HCl 1,5M, pH 8,8 0,6 ml 1,9 ml 1,9 ml

Acrylamide 30% 0,4 ml 2 ml 2,5 ml

SDS 20% 0,15 ml 0,15 ml 0,15 ml

APS 10% 13 µl 30 µl 30 µl

TEMED 3 µl 5 µl 5 µl

3.5.9. Western blotting and protein detection

Proteins subjected to SDS-PAGE were electroblotted onto a Hydrobond-C nitrocellulose membrane (Amersham) using a semi-dry blotting system (Multiphor II) with blotting buffer at 1mA/cm² for 1h. Nonspecific binding sites were blocked with 5% non-fat dried milk (NFDM) freshly made in TBS/Tween rocked on the orbital shaker for 1h.

Afterwards blots were rinsed 3 times in TBS/Tween and incubated with the primary antibody, diluted in the TBS + 1% NFDM, overnight at 4°C on the rotating shaker (antibodies and dilution factors are listed in the table 7). On the next day membranes were washed 3 times with TBS/Tween for 10 min and incubated with horseradish peroxidase-conjugated secondary antibody, diluted in TBS/Tween for 1h at room temperature. For visualization, the membrane after washing with TBS/Tween (3x5min) was treated with SuperSignal West Pico or SuperSignal West Femto (used only for nitrotyrosine detection) chemiluminescent substrate (both from Pierce)according to the

manufacturer's instructions. Image capture was performed using a LAS-1000 Luminescence Image analyser (Fuji Film). Obtained images were analyzed using the Aida Image Analyzer software, version 3.22 (Raytest).

Table 7. Antibodies that were used for protein immunodetection on Western blots

primary antibody dilution factor secondary antibody dilution factor iNOS (from Prof. Pfeilschifter) 1:500 GAR-POX (Dianova) 1:50 000

iNOS (Cayman, 160862) 1:800 GAR-POX 1:50 000

COX-2 (Cayman, 160116) 1:500 GAR-POX 1:30 000

nitrotyrosine

(Cayman, 189 542) 1:250 GAM-POX (Dianova) 1 :30 000

PCS 3 ( Dr. Thomas Klein) 1:800 GAR-POX 1:50 000

k376 (Dr. Thomas Klein) 1:250 GAR-POX 1:50 000

3.5.10. Stripping of Western blots

Bound antibody was removed from Western Blot membranes by incubation in Western blot stripping buffer for 30 min at 60°C. Afterwards membranes were washed three times in TBS and blocked with 5% BSA/TBS for 30 min. After this procedure Western blots were ready for another immunodetection of proteins.

3.5.11. Two-dimensional gel electrophoresis 3.5.11.1. Cell extract.

Primary human chondrocytes after isolation were grown in plastic flasks in DMEM/F12 20%FCS for three days. Confluent cells were incubated with IL-1β 0,5nM or SIN-1 250µM in DMEM/F12 10%FCS w/o phenol red. After 24h Griess assay was performed in cell culture supernatant to test the stimulation. The medium was collected and the cells were washed with PBS, loosened by trypsin/ethylenediaminetetraacetic acid (EDTA) and centrifuged (5min, 1200rpm,RT). Afterwards the cells were washed with TBS and TBS/distillated water (50/50%). Then a lysis buffer [7 M Urea, 2M Thiourea, 4% CHAPS, 1% DTT; 2% IPG-ampholytes] was added to the cell pellets.

Further analysis (two-dimensional gel electrophoresis and protein identification) was performed at ALTANA Pharma in the department RDR/PX (Head of the Department Dr.

Sascha Dammeier).

3.5.11.2. Two-dimensional gel electrophoresis

Two-dimensional gel electrophoresis was performed with the IEF system (Amersham Biosceineces; Multiphor II Sytem). The first dimension used 7-cm nonlinear pH 3–10, immobilizedpH gradient (IPG) strips loading 40 µg protein, or 7-cm nonlinear pH 6,2-7,5 strips loading 100µg protein (for COX-2).

IPG strips were rehydrated over night as described in the supplier’s manual and then isoelectric focusing was performed using following programs:

IPG strips pH 3-10:

Step V mA W Time (h) Vh 1 0 - 200 2 5 0,01 1 2 200 - 3500 2 5 1,3 2405

3 3500 2 5 1,05 3675

total: 2,36 h 6081 Vh IPG strips pH 6,2-7,5:

Step V mA W Time (h) Vh 1 0 - 200 2 5 0,01 1 2 200 - 3500 2 5 1,3 2405

3 3500 2 5 5,00 17500

total.: 6,3 h 19,9Vh

For the second dimension, the IPG strips were equilibrated 12 to 15 min in IPG strip reducing buffer and then alkylated for 5 min with JAA in IPG strip alkylating buffer.

IPG strips were placed on top of the 12% SDS-PAGE gel and covered with melted agarose in Laemmli buffer.

The second dimension SDS-PAGE was performed essentially according to Laemmli.

After completion of the run, the acrylamide gels were soaked in transfer buffer (20 mM

Tris-HCl, 96 mM glycine, and 20% methanol) and then partially transferred onto a polyvinylidene difluoride (PVDF) membrane by

using a wet transfer apparatus as suggested by the manufacturer.

The gels were then stained with colloidal Coomassie blue (Roti Blue stain; Roth), and Western blot analysis was performed on the PVDF membrane.

3.5.11.3. Western blot analysis.

PVDF membranes were blocked for 2h by using a blocking buffer [20 mM Tris, 150 mM NaCl (pH 7.5), 0.1% Tween-20, and 5% BSA]. Membranes then were probed overnight at 4°C with a monoclonal antibody against nitrotyrosine (1:5,000; clone 1A6, Upstate Biotechnology; Lake Placid, NY) in blocking buffer. The membranes then were washed four times in washing buffer [20 mM Tris, 150 mM NaCl (pH 7.5), and 0.1%Tween-20]

for 15 min each wash. The membranes were then probed with a goat anti-mouse antibody (horseradish peroxidase conjugate, 1:50,000). After the membranes were washed four times in a washing buffer, the immunopositive spots were visualized by using LumiLight-Plus (Roche) as directed by the manufacturer. In experiments where samples needed to be compared, the membranes were blotted on the same membrane and exposed simultaneously.

3.5.11.4. Protein identification (MALDI-TOF).

Proteins were identified by matrix-assisted laser desorption ionization - time of flight (MALDI-TOF) mass spectrometric analysis of in-gel tryptic digests of immunopositive spots. The two-dimensional gel spots were excised and cut into ca. 1mm³ cubes, and Coomassie blue was washed away using following protocol:

• distilled water MilliQ, ~50µl, 15min, RT

• 50% acetonitrile in 50 mM ammonium bicarbonate 15min, RT, shaking

• 50% acetonitrile in 15 mM N-ethylmorpholine till gel pieces are shrunken, then acetonitrile removed

• rehydration in 50 mM ammonium bicarbonate ~50µl, 5min

• 50% acetonitrile ~50µl, 15min

• the excess of fluid removed

• 50% acetonitrile till gel cubes are shrunken, then acetonitrile removed

• dried in Speed Vac, ~5min.

After being destained and dried the gel pieces were rehydrated in 20-25µl ammonium bicarbonate containing 2,5ng/ml trypsin and incubated overnight at 37°C. The in-gel tryptic digest was extracted with 50% acetonitrile containing 1% trifluoroacetic acid. The extract was dried and after resolving in 0.2 % TFA desalted using Zip-Tipµ-C18

(Millipore). The desalted extract was subjected to MALDI-TOF mass spectrometric analysis by using a Bruker Ultraflex instrument in reflector mode equipped with a nitrogen laser (337 nm). α-Cyano-4-hydroxy-cinnamic acid was use as Matrix. The elution solution (1µl of CH3CN/0.2 % TFA 2/1) from Zip-Tipµ-C18 desalting was directly spotted on a thin layer of the matrix. Each spectrum was accumulated for ca. 300 laser shots. Measured peptide masses were used to search with Mascot the MSDB database for protein identifications.

3.6. Staining of cartilage and chondrocytes

3.6.1. Preparation of cartilage sections

The cartilage was dissected from the underlying bone and fibro-cartilaginous areas were discarded. Cartilage samples were fixed with 7,5% paraformaldehyde in PBS pH 7.4 and stored at 4° for min. 1 week. After fixation the specimens were washed with tap water for 2 hours and then decalcified in TBD-2 solution, which is a formic acid solution.

The decalcifier solution was replaced every 24h. Using this protocol cartilage sections were fully decalcified in 4-7 days.

To remove decalcifier from the tissue, cartilage specimens were washed for 12 h in tap water, which was frequently changed.

Decalcified cartilage was dehydrated in a graded series of alcohols (70% 2-propanol: 2h 15 min, 80% 2-propanol: 2h 15min, 90% 2-propanol: 2h 15min, 100% 2-propanol: 2h 15min, 100% 2-propanol: 2h 15min, 100% 2-propanol: 2h 15min, 100% 2-propanol: 2h 15min, xylol: 2h, xylol: 2h, xylol: 2h) and embedded in low-melting-point paraffin (paraffin 60°C: 3h, paraffin 60°C: 3h) using a Shandon Citadel 2000 tissue processor.

Tissue sectioning:

The paraffin blocks were placed in a microtome and trimmed down to the cartilage surface, then 4 or 5 µm cartilage sections were cut. Tissue sections were floated in a water bath at 46°C and collected on Histo Bond glass slides. Afterwards glass slides with tissue sections were dried for 1-2 h at 60°C.

3.6.2. Histochemical and fluorescence staining of tissue samples

Tissue sections were deparaffinized in xylene, passed through decreasing gradations of ethanol (100%, 80%, 50%), and washed in TBS. To enhance protein antigen accessibility, the sections were incubated with digestive enzymes. For each antibody four digestion protocols were tested (all in humid chambers):

1. hyaluronidase (2mg/ml PBS, pH 5,5; 15 min at 37°C) and then pronase (1mg/ml PBS, pH 7,5; 30 min at 37°C),

2. protease (0,02mg/ml TBS, pH 7,6; 60 min at 37°C),

3. chondroitinase ABC (0,05U/ml in 0,1M Tris-acetat buffer, pH 7,6; 30 min at 37°C),

4. trypsin-EDTA (the same as for the cell culture, 60min at 37°C).

Enzymes were removed with three 2 min washes with TBS or PBS and 5% BSA in PBS for 30min was placed on the sections to block the non-specific binding. Afterwards the sections were incubated with primary antibodies. Primary antibodies were diluted with TBS containing 1% BSA immediately prior to use and placed on the sections, followed by overnight incubation in humid chamber at 4°C. After washing with TBS to remove residual primary antibody, secondary antibody was used to visualise a presence of examined protein:

For immunohistochemical staining: the sections were incubated with biotin conjugated secondary antibodies for 30 min in a humid chambers, followed by washes and incubation with streptavidin-avidin-biotin- alkaline phosphatase complex (StreptABComplex/AP, DAKO) for 30 min at room temp. Finally substrate reagent: Fast Red from Sigma was added. After 15-30min reaction sections were washed in distilled water and mounted with AquaTex.

For immunofluorescence staining: the sections were incubated with Cy³ or Cy² conjugated secondary antibody for 30 min at room temp. in a humid chamber. Finally, nuclei were stained with Hoechst (1:10 000, 5 min) and the sections were mounted with Gel Mount.

Sections were observed and photographed using a DMLB fluorescence microscope from Leica, pictures were recorded, and if necessary overlayed, using IM1000 software from Leica.

3.6.3. Cytospin preparations

Chondrocytes from digested cartilage, dissolved alginate beads or from trypsinized monolayer culture were dispersed by repeated pipetting to obtain a single-cell suspension in PBS. 100µl of cell suspension were placed in cytofunnel chamber and centrifuged for 3 min, 1500rpm at medium acceleration. Cytoslides were then air-dried for 1h, afterwards fixed in 100% methanol for 3min, washed with PBS and blocked with 5% BSA in PBS for 30min. Further staining was performed to the same protocol as cartilage sections.

3.6.4. Evaluation of antibodies 3.6.4.1. iNOS TK2553

Paraffin embedded cartilage: the best results were obtained with chondroitinase digestion.

For all immunostainings: 1:100 dilutions in 1%BSA/TBS, as secondary antibody:

- immunofluorescence: Cy³ Goat anti-Rabbit (Dianova 111-165-144) diluted in PBS 1:800.

- immunohistochemistry: Biotin-SP-conjugated Affini Pure Donkey Anti-Rabbit IgG (Dianova 711-065-152) diluted in TBS 1:1000

3.6.4.2. β-actin / Sigma A5441 clone AC-15

For immunofluorescence: 1:500 dilutions in 1%BSA/PBS, as secondary antibody:

- Cy² Goat anti-Mouse (Dianova 115-225-146) diluted in PBS 1:200.

3.6.4.3. Nitrotyrosine

Anti-Nitrotyrosine (rabbit immunoaffinity purified IgG) / Upstate Cat. # 06-284

- immunofluorescence (cytospin): dilution: 0,5µg/ml in 1%BSA/PBS, as secondary antibody Cy² Goat anti-Rabbit (Dianova 111-225-144) diluted in PBS 1:800.

- immunohistochemistry (tissue): dilution: 5µg/ml in 1%BSA/TBS, as secondary antibody Biotin-SP-conjugated Affini Pure Donkey Anti-Rabbit IgG (Dianova 711-065-152) diluted in TBS 1:1000. Paraffin embedded cartilage: best results obtained with protease or hyaluronidase/pronase digestion.

Anti-Nitrotyrosine, clone 1A6 / Upstate Cat. # 05-233

- immunofluorescence (cytospin): dilution: 1µg/ml in 1%BSA/PBS, as secondary antibody Cy² Goat anti-Mouse (Dianova 115-225-146) diluted in PBS 1:200.

Anti-Nitrotyrosine, Monoclonal Antibody / Cayman Cat. # 189542

- immunofluorescence (cytospin): dilution: 0,5µg/ml in 1%BSA/PBS, as secondary antibody Cy² Goat anti-Mouse (Dianova 115-225-146) diluted in PBS 1:200.

- immunohistochemistry (tissue): dilution: 10µg/ml in 1%BSA/TBS, as secondary antibody Biotin-SP-conjugated Affini Pure Donkey Anti-Mouse IgG (Dianova 715-065-150) diluted in TBS 1:200. Paraffin embedded cartilage: the best results obtained with trypsin or chondroitinase digestion.

The best results for nitrotyrosine staining by immunofluorescence technique were

The best results for nitrotyrosine staining by immunofluorescence technique were

Im Dokument The Role of Nitric Oxide in Chondrocyte Models of Osteoarthritis (Seite 45-0)