UM-SCC 46 cells were plated in Lab-TekR II chamber slide (Life Technologies) at 15,000 cells per well in 500μl complete media. Upon achieving 70 to 80% cell confluent, NIK inhibitor (1, 3[2H, 4H]-Isoquinolinedione) was added to individual wells followed by LTβ (100ng/ml) stimulation for another 4 or 12hours. Cells were then fixed using ice cold methanol for 15minutes, and permeabilized on ice (0.5% Triton X-100 and 0.05% SDS). Then cells were blocked on ice for 1 hour using blocking solution (0.1% Tween-20 and 3% BSA). Anti-RELB antibody (Santa Cruz) or Anti- NIK antibody (abcam) was added to each well at 1:100 dilution and incubated for 1 hour at room temperature. Cells were incubated with AF-594-linked IgG (1:1000 dilution) for 45 minutes in dark. The slides were mounted with DAPI VECTASHIELD mounting media, and were visualized on LSM 780 confocal microscope. Confocal images were analyzed using Zen 2012 SP1 software (black and blue editions).
Anti nik antibody
Manufactured by Abcam
Anti-NIK antibody is a laboratory reagent used to detect and study the expression of the NIK (NF-kappa-B-inducing kinase) protein in biological samples. NIK is a key regulator of the non-canonical NF-kappa-B signaling pathway, which plays a role in various cellular processes. This antibody can be used in techniques such as Western blotting, immunohistochemistry, and immunoprecipitation to investigate the presence and localization of NIK in cells and tissues.
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Lab products found in correlation
Alexa fluor 488 conjugated anti mouse igg antibody, by Thermo Fisher Scientific (1 mentions)
Anti ki67 antibody sp6, by Abcam (1 mentions)
Eclipse ti inverted microscope, by Nikon (1 mentions)
Hoechst 33342, by Merck Group (1 mentions)
Deadend colorimetric tunel system, by Promega (1 mentions)
Fv1000 confocal microscope, by Olympus (1 mentions)
2 protocols using anti nik antibody
1
Visualizing NIK and RELB Localization
2
Immunofluorescence and Immunohistochemical Analysis
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For immunofluorescence staining, fixed and permeabilized cells were stained with an anti-DSG1 antibody (129204, R&D Systems), followed by an Alexa Fluor 488-conjugated anti-mouse IgG antibody (Invitrogen). Nuclei were visualized by Hoechst 33342 (Sigma-Aldrich) staining. An FV1000 confocal microscope (Olympus) and accompanying FV10-ASW software were used to acquire confocal images. For immunohistochemical staining, fixed and paraffin-embedded tumor sections were stained with hematoxylin and eosin (HE), anti-Ki-67 antibody (SP6, Abcam), and anti-NIK antibody (Abcam), using standard procedures. TUNEL staining was performed with the DeadEnd Colorimetric TUNEL System (Promega). Images were captured with an Eclipse Ti inverted microscope (Nikon) and accompanying NIS-Elements software. Quantification was performed using ImageJ software. The number of Ki-67+ or TUNEL+ cells in each field was counted. NIK expression in tumor cells was scored (intensity: 0 = negative, 1 = weak, 2 = moderate, 3 = strong), and the H score was calculated using the following equation: H score = 1 × (% of cells with intensity 1) + 2 × (% of cells with intensity 2) + 3 × (% of cells with intensity 3).
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