Dako dual endogenous enzyme blocking reagent

24 hours after hydrogel encapsulation or pellet culture formation, cells were stained using the LIVE/DEAD cell viability assay (Life Technologies). Samples were imaged using fluorescent confocal laser scanning microscopy (FV1000 Olympus). Cell viability was assessed using computer-assisted, colorimetric-based quantification methods.
Additionally, 5 µm sections of frozen cell-laden hydrogels and pellets were immunostained using a goat polyclonal anti-N-cadherin antibody (#sc-31030, Santa Cruz Biotechnology, Inc., Dallas, TX) and an immunoperoxidase detection kit (Vectastain ABC Kit, Vector Laboratories, Burlingame, CA). Frozen samples were warmed to room temperature, quenched of endogenous peroxidase activity (DAKO Dual Endogenous Enzyme Blocking Reagent; DAKO S2003), and blocked with Vectastain 5% normal rabbit serum (NRS). Slides were then incubated with a 1:50 dilution of goat polyclonal anti-N-cadherin antibody in 2.5% NRS in phosphate buffered saline with Tween-20 (PBST) overnight at 4 °C. Slides were washed and then incubated in Vectastain biotinylated rabbit anti-goat secondary antibody in PBST, washed, and incubated with Vectastain ABC reagent. Slides were developed using the Vectastain ImmPACT DAB peroxidase reagent and counter-stained for 1 min using haematoxylin (Invitrogen).

Intact left knee joints were fixed in neutral buffered formalin for 72 hrs, washed and decalcified in 5% formic acid (Immunocal, Decal Chemical Corp) and then paraffin embedded, and sectioned. Sections were baked overnight at 60°C, deparaffinized and rehydrated through graded ethanol solutions. For immunohistochemical analyses, antigen retrieval was performed in 10% hyaluronidase (MP Biomedicals) in PBS for 10 min at 37°C. Endogenous peroxidase was quenched with DAKO Dual Endogenous Enzyme Blocking Reagent for 30 min (Dako). Sections were blocked with normal goat serum (Vectastain Elite Rabbit IgG Kit) for 30 min, and incubated overnight at 4°C with a specific primary OGR1 antibody (LifeSpan BioSciences, Inc.) 1:200 in 2% normal goat serum. Binding was detected with a biotinylated goat anti-rabbit (Vectastain Elite Rabbit IgG Kit) secondary antibody and subsequent incubation with Vectastain ABC Reagent. The color reaction was detected with Vector Impact DAB (Vector SK-4105) and counterstained with hematoxylin (Zymed).