Mouse eyes were collected, fixed with 4% paraformaldehyde, and processed for paraffin-embedded sectioning after 24 weeks of the food regimen. Paraffin sections (4 µm) of the mouse eyes from all four experimental groups were mounted on the same slide. After deparaffinization, sections were permeabilized in citrate buffer (pH 6.0) at 80 °C for 1 hour. Sections were then blocked with 10% goat serum for 2 hours at room temperature and incubated with anti-phospho extracellular signal-regulated kinase (ERK) (Cat.# 4370, Cell Signaling, Danvers, Massachusetts, USA) or anti-phospho NFĸB P65 (Cat.# 3033, Cell Signaling) overnight at 4 °C. After washing with PBS, sections were incubated with Alexa Fluor 488/568 goat anti-rabbit IgG (1:50 dilution; Molecular Probes/Life Technologies/Thermo Fisher Scientific, Grand Island, New York, USA) for 2 hours at room temperature and mounted with ProLong Gold antifade reagent containing 4′,6′-diamidino-2-phenylindole (Invitrogen/Life Technologies/Thermo Fisher Scientific). Images were obtained using a Zeiss Stallion digital imaging workstation equipped with a Zeiss Axiovert 200M microscope (Carl Zeiss Microscopy) under identical settings. The average fluorescence intensity was measured in the outer and inner segments of photoreceptors by ImageJ (National Institutes of Health, Bethesda, Maryland, USA).
Anti phospho nf b p65
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-phospho NFĸB P65 is a primary antibody that specifically recognizes the phosphorylated form of the NFĸB p65 subunit. NFĸB p65 is a key component of the NF-κB transcription factor complex, which plays a crucial role in regulating immune and inflammatory responses.
Automatically generated - may contain errors
Lab products found in correlation
Anti nf bp65, by Cell Signaling Technology (2 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (1 mentions)
Axiovert 200m microscope, by Zeiss (1 mentions)
Supersignal west pico femto chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Anti elk1, by Cell Signaling Technology (1 mentions)
Anti β actin, by Cell Signaling Technology (1 mentions)
Anti 1 b α, by Cell Signaling Technology (1 mentions)
Anti gapdh antibody, by Abcam (1 mentions)
Anti gsk3β, by Cell Signaling Technology (1 mentions)
Anti axin2, by Cell Signaling Technology (1 mentions)
Anti cyclin d1, by Santa Cruz Biotechnology (1 mentions)
Anti phospho jnk, by Cell Signaling Technology (1 mentions)
3 protocols using anti phospho nf b p65
1
Immunohistochemical Analysis of Retinal Signaling
2
Western Blot Analysis of Cellular Signaling
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Samples for Western blots were collected, prepared and analysed as described previously.38 Briefly, 661W cells were harvested and lysed in a Tris lysis buffer (in mM): 50 Tris, 1 EGTA, 150 NaCl, 1% Triton X‐100, 1% β‐mercaptoethanol, 50 NaF, and 1 Na3VO4, pH 7.5. Samples were separated on 10% sodium dodecyl sulphate‐polyacrylamide gels by electrophoresis and transferred to nitrocellulose membranes. The membranes were blocked in 3% BSA in Tris‐buffered saline, Tween 20 (TBST) at room temperature for 1 h and incubated in primary antibodies overnight at 4°C. After washing with TBST, the membranes were incubated in anti‐rabbit IgG HRP‐linked secondary antibody (1:1000, #7074S, Cell Signaling) at room temperature for 1 h. The blots were visualized using Super Signal West Pico/Femto chemiluminescent substrate (#34078 / #34096, ThemoFisher). Band intensities were quantified using ImageJ (NIH). The primary antibodies used in this study were as follows: anti‐ELK1 (1:500, #9182S, Cell Signaling), anti‐phospho NFĸB P65 (1:1000, #3033, Cell Signaling), anti‐NFĸB P65 (1:1000, #8242S, Cell Signaling) and anti‐β‐actin (1:2000, #8457L, Cell Signaling). The band intensities of ELK1 were normalized to those of β‐actin. The band intensities of phosphorylated NFĸB P65 (pP65) were normalized to those of total NFĸB P65 (P65) and β‐actin.
3
Western Blot Analysis of Wnt Signaling
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Western blottings were performed as described [45 (link)]. Antibodies used included anti-phospho-β-Catenin (Ser33/Ser37/Thr41), anti-GSK-3β, anti-Axin-2, anti-phospho-JNK, anti-NF-ĸB/p65, anti-phospho-NF-ĸB/p65 and anti-IĸBα, which were purchased from Cell Signaling Technology, anti-Cyclin D1 (Santa Cruz) and anti-GAPDH antibody (Abcam). Specific bands were visualized with the West Dura Extended Duration Substrate (Pierce) and Chemiluminescence Analysis System (ChemiScope Series, Clinx). The cropped blots were displayed in the figures. The intensities of bands were measured by the ChemiScope Analysis System.
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