Paraffin-embedded tissue sections were stained immunohistochemically with antibodies against Ki67 (clone TEC-3, DAKO), p65-pho (Abcam) and AR-V7 (Precision). The primary antibody was incubated at the appropriate concentration (1:1000) for one hour at room temperature. The secondary antibody was incubated for 60 minutes. Slides were rinsed extensively in tap water, counterstained with Mayer’s hematoxylin and mounted. For quantitation of the cell proliferation, the cells were counted as positive for Ki67 when nuclear immunoreactivity was observed. Each tissue section was counted manually in three different areas to assess the Ki67 positive cells index. The data were then presented as number of Ki67 positive cells/400x microscope field. Expression of p65-pho and AR-V7 were measured by calculating the percentage of DAB-stained nuclear area over total nuclear area (labeling index) using ImmunoRate program.61
P65 pho
Manufactured by Abcam
P65-pho is an antibody that detects phosphorylated NF-κB p65 subunit. It can be used in various applications to study the activation of the NF-κB signaling pathway.
Automatically generated - may contain errors
4 protocols using p65 pho
1
Immunohistochemical Analysis of Cell Proliferation and Protein Expression
2
Immunohistochemical Staining of Prostate Tissues
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Paraffin-embedded tissue sections were stained immunohistochemically with antibodies against GRP-R (Abcam), Ki67 (clone TEC-3, DAKO), AR (N-20, Santa Cruz), p65-pho (Abcam) and AR-V7 (Precision). The primary antibody was incubated at the appropriate concentration (GRP-R, 1:200; Ki67, 1:1000; AR, 1:1000; p65-pho, 1:1000; AR-V7, 1:200) for one hour at room temperature. The secondary antibody was incubated for 60 minutes. Slides were rinsed extensively in tap water, counterstained with Mayer's hematoxylin and mounted.
3
Immunohistochemical Analysis of Cell Proliferation and Protein Expression
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Paraffin-embedded tissue sections were stained immunohistochemically with antibodies against Ki67 (clone TEC-3, DAKO), p65-pho (Abcam) and AR-V7 (Precision). The primary antibody was incubated at the appropriate concentration (1:1000) for one hour at room temperature. The secondary antibody was incubated for 60 minutes. Slides were rinsed extensively in tap water, counterstained with Mayer’s hematoxylin and mounted. For quantitation of the cell proliferation, the cells were counted as positive for Ki67 when nuclear immunoreactivity was observed. Each tissue section was counted manually in three different areas to assess the Ki67 positive cells index. The data were then presented as number of Ki67 positive cells/400x microscope field. Expression of p65-pho and AR-V7 were measured by calculating the percentage of DAB-stained nuclear area over total nuclear area (labeling index) using ImmunoRate program.61
4
Western Blot Analysis of Protein Expression
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Whole cell lysate was extracted from experimental cells. A 20μg aliquot of each protein sample was separated on a 4 to 12% Tris-glycine gradient gel (NOVEXTM), and then transferred to nitrocellulose membranes (Schleicher & Schuell, Germany). The membranes were blocked with 5% skim milk in TBS-T (Trypsin buffered saline, 1% Tween-20) buffer. The AR (N-20, Santa Cruz; 1:1000), p65-pho (Abcam; 1:300) or GRP-R (Abcam; 1:200) antibody was added and the blots were incubated o/n in 4 C°. After washing three times for 10 minutes each in TBS-T, incubation was performed for 1 hour with the secondary horseradish-peroxidase-conjugated anti-rabbit/anti-mouse antibody. Actin was used as the loading control. The signals were developed by an ECL detection system (Amersham Biosciences, Amersham, USA).
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