Anti nf κβ sc 8008

Human articular chondrocytes were cultured on chamber slides and fixed with 4% formaldehyde (PFA) for 10 min at room temperature. Frozen cartilage sections were sequentially sectioned (4 µm) and processed as previously described with minor variations [45 (link)]. The samples were counterstained with haematoxylin–eosin or DAPI (Sigma Aldrich, Darmstadt, Germany). Anti-Podoplanin (18H5) antibody was supplied by Merck Millipore and anti-NF-κβ (sc-8008) was supplied by Santa Cruz Biotechnology. Negative controls without primary antibody were performed to test the specificity of each antibody. haematoxylin–eosin, Safranin-O Fast Green, Masson’s trichrome, and toluidine blue were used to stain the cartilage sections. Images were taken on an Olympus BX61 microscope using a DP71 digital camera (Olympus); the AnalySIS^D 5.0 software (Olympus Biosystems, Hamburg, Germany) was used for image calibration and quantification.

Sections (5 µm) were cut for immune staining. The tissue sections were incubated with primary antibodies of IL-1 β, TNF- α, NF-κβ, and VEGF (anti-IL-1β (SC-12742), anti-TNF-α (SC-52746), anti-NF-κβ (SC-8008), and anti-VEGF (SC-57496), Santa Cruz, Biotechnology, Inc., at a dilution of 1:200) for 1 h at room temperature, followed by washing with TBS. Endogenous peroxidases were blocked using hydrogen peroxide. Afterward, an HRP-labeled detection kit (Bio SB, BSB0001, USA) was used as per the manufacturer’s instructions to develop the positive reaction. Negative controls were obtained by deleting incubation with primary antibodies. Positive immune staining was quantified as area percent using LAS-X software (Leica v4.13, Germany).