Dual endogenous enzyme block

Immunohistochemical staining of tumor tissue samples from patients with adenocarcinoma was conducted as previously described. Briefly, sections for analysis of Slug or MDA-9/Syntenin protein expressions were first autoclaved in Trilogy Solution (Cell Marque Corp) or Antigen Retrieval Citra Solution (Biogenex, San Ramon, CA) at 121°C for 10 min. The samples were subsequently treated with 3% H₂O₂-methanol and incubated with DakoCytomation Dual Endogenous Enzyme Block (DakoCytomation) for 10 min, Ultra V Block (Lab Vision Corporation) for 10 min, antibody-dilution buffer (Ventana Medical Systems, Inc., Tucson, AZ) for 10 min, and with anti-Slug (1:75, ABGENT) antibody at room temperature or the anti-MDA-9/Syntenin antibody (1:100, Santa Cruz Biotechnology) overnight at 4°C.
Immunostaining was detected using a Super Sensitive Non-Biotin Polymer HRP Detection System (BioGenex, San Ramon, CA) according to the manufacturer's protocol.

Paraffin-embedded tissue sections were deparaffinized and rehydrated with graded ethanol dilutions. Antigen retrieval was applied to the tissue sections in a pressure cooker using an Antigen Retrieval Machine (2100 Retriever) with EDTA Buffer pH 9. Immunohistochemistry was performed using the Dako REAL Detection Kit System, Alkaline Phosphatase/RED, Rabbit/Mouse Kit (Dako Cat.# S2022, distributed by Agilent Technologies AG) according to the manufacturer’s instructions and as previously described [78 (link)]. For washing steps, the Dako washing buffer diluted with 1:10 with distilled water was used, and the Dako dual endogenous enzyme block (S2003) was applied to the tissue section before staining to inhibit endogenous phosphatase activity. Tissue sections were incubated with mouse monoclonal anti-human 4-HNE (1:50 dilution; clone 12F7, Invitrogen cat. #MA5-27570, distributed by ThermoFisher Scientific) in Dako antibody diluent S2022, followed by a biotin-streptavidin system coupled with alkaline phosphatase and counterstained with Hematoxylin dye (Dako Cat. #S2020) and mounted with Aquatex (Merck Cat. #1.08562.005, distributed by Sigma-Aldrich, Buchs, Switzerland). The images were visualized using a light microscope (Carl Zeiss Micro Imaging, Jena, Germany).

Immunohistochemical staining was performed on serial formalin-fixed and paraffin- embedded tissues sectioned at 4 μm-thickness as we previously described [40 (link)]. Tissue sections were deparaffinized by two consecutive incubations in xylene for 10 min each, followed by rehydration through two changes of absolute ethanol, graded decreasing concentrations of ethanol for 5 min each and finally in distilled water. For antigen retrieval, slides were incubated in citrate buffer (pH = 6.0) in a water steamer for 30 min. Slides were left to cool at room temperature for 20 min then washed 3 × 5 min with PBS. Endogenous peroxidase activity of the tissue was blocked with 3% hydrogen peroxide for 5 min (Dual Endogenous Enzyme block, Dako K4065, Glostrup, Denmark) and slides were washed with PBS 3 × 5 min. Tissue sections were blocked in 1% BSA/PBS and incubated overnight at 4 °C in a humidified chamber with the primary anti-CD44 (dilution 1:800) and anti-Syndecan-1 antibodies (dilution 1:100). Afterwards, slides were washed 3 × 5 min and incubated with HRP-Rabbit/Mouse (DAKO EnVision + Dual Link System-HRP (DAB+) for 30 min at room temperature. Then, nuclei were counterstained with hematoxylin, sections were mounted with Permount® and imaged. Negative control slides were run in parallel where primary antibodies were omitted.

Paraffin-embedded decalcified longitudinal sections of either 3- or 11-week old femurs were deparaffinized and hydrated. Von kossa staining was performed as described previously⁹¹ . For IHC, deparaffinized and hydrated sections were incubated for 1 h at 37 °C with ABCase for Bgn or keratanase for Fmod (Seikagaku biobusiness corp.; Japan), following antigen retrieval (Unitrieve, Innovex), and quenching of endogenous peroxidase activity with dual endogenous enzyme block (Dako), sections were blocked with 10% normal goat serum for 1 h at 37 °C. Bgn rabbit antisera, LF-159 (1:500), or Fmod rabbit antisera LF-150 (1:500; both from Dr. Larry W. Fisher, NIH) [Figure S1] were added to sections and incubated overnight at 4 °C. The samples were then incubated with Super PicTure™ Polymer detection kit (Invitrogen) for 10 min at room temperature and detected with ImmPACT™ AEC (Vector laboratories). Mayer’s hematoxylin was used as counterstain. Slides were scanned using an Aperio ScanScope slide scanner.