The kidneys were rapidly removed from each mouse. The tissues were immediately fixed in 4% paraformaldehyde and embedded in paraffin. Serial (4 μm thick) sections were deparaffinized in xylene, rehydrated using descending grades of ethanol, and stained with hematoxylin and eosin (H&E) and periodic acid-Schiff (PAS). Immunohistochemical staining was performed using an anti-nuclear factor kappa-B (NF-κB) p65 primary antibody (Santa Cruz Biotechnology, Dallas, TX, USA), followed by incubation with horseradish peroxidase-conjugated anti-rabbit IgG secondary antibody (Santa Cruz Biotechnology). Images were captured using an Olympus BX53 microscope (Tokyo, Japan). Tubular damage in PAS-stained kidney sections was scored according to the level of cortical tubular injury as previously described: 0, normal; 1, 1–10%; 2, 11–25%; 3, 26–45%; 4, 46–75%; and 5, 76–100% [12 (link)]. The number of NF-κB-activated cells in each section was determined by counting the number of cells showing nuclear p65-positive staining in 10 fields per slide (×200).
Nf κb p65 primary antibody
Manufactured by Santa Cruz Biotechnology
Sourced in United States
The NF-κB p65 primary antibody is a laboratory reagent used to detect and study the p65 subunit of the NF-κB transcription factor. NF-κB is a key regulator of immune and inflammatory responses in cells.
Automatically generated - may contain errors
Lab products found in correlation
Goat serum, by Solarbio (2 mentions)
Horseradish peroxidase conjugated anti rabbit igg secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Bx53 microscope, by Olympus (1 mentions)
Total protein extraction kit, by Boster Bio (1 mentions)
Gs 710, by Bio-Rad (1 mentions)
Hrp labeled streptavidin, by Beyotime (1 mentions)
A microscope, by Olympus (1 mentions)
Hematoxylin, by Solarbio (1 mentions)
3 3 diaminobenzidine (dab), by Solarbio (1 mentions)
Gfap primary antibody, by Santa Cruz Biotechnology (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Biosharp (1 mentions)
Fluorescence microscope, by Olympus (1 mentions)
Cy3 labeled goat anti mouse igg h l, by Beyotime (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
5 protocols using nf κb p65 primary antibody
1
Kidney Histopathological Analysis
2
Quantifying NF-κB p65 Protein Levels
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Total proteins were purchased from pancreatic tissue using a total protein extraction kit (Boster, Wuhan, China). Protein concentrations were determined using a commercial kit (Pierce, Rockford, IL, USA). Each 20-μg aliquot of total protein was loaded onto sodium dodecyl sulfate-polyacrylamide gel for electrophoresis and then transferred onto membranes. Following complete protein transfer, the membranes were blocked with 5% milk powder solution for 2 h and incubated with NF-κB p65 primary antibody (Santa Cruz Biotechnology, Inc., Dallas, TX, USA) overnight. After washing the membranes, the secondary antibody (Boster, Wuhan, China) was applied and incubated for 2 h at room temperature (RT). Bands were quantified by a calibrated imaging densitometer (GS-710; Bio-Rad) and analyzed by “Quantity One” software (Bio-Rad). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal reference.
3
Naringin Modulates NF-κB p65 in Rat Retina
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IHC assay was performed to detect the effects of naringin on expression of NF-κB p65 in rat retinal tissues. Sample sections (5 μm) were deparaffinized with xylene and rehydrated with ethanol. Antigen retrieval was performed with sodium citrate buffer in a microwave for 10 min. The sections were added with 3 % H2O2 for 15 min at room temperature (RT). Thereafter, the sections were blocked with goat serum (Solarbio) for 15 min at RT. After serum deprivation, the sections were incubated with NF-κB p65 primary antibody (1:50; Santa cruz, Santa Cruz, CA, USA) at 4 °C overnight and then with a Biotin-labeled Goat Anti-Rabbit IgG(H+L) secondary antibody (1:200; Beyotime, Beijing, China) at 37 °C for 30 min. Then the sections were incubated with HRP-labeled streptavidin (Beyotime) at 37 °C for 30 min and color was developed using DAB (Solarbio). The sections were counterstained with hematoxylin (Solarbio). The positive staining was visualized using a microscope (Olympus).
4
Immunofluorescence Analysis of Retinal GFAP and NF-κB
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IF was carried out to analyze glial fibrillary acidic protein (GFAP) expression in rat retinal tissues and the distribution of NF-κB p65 in rMC1 cells. The retinal sections embedded in paraffin were deparaffinized before antigen retrieval. The rMC1 cells seeded on glass cover slips were treated with 4% paraformaldehyde for 15 min and then permeabilized with 0.1% tritonX-100 for 30 min. The tissue sections or cell slides were blocked with goat serum (Solarbio) and incubated with GFAP primary antibody (1:100; Santa cruz) or NF-κB p65 primary antibody (1:200; Santa cruz) at 4 °C overnight. The secondary antibody Cy3-labeled Goat Anti-Mouse IgG(H+L) (1:500 for tissue sections or 1:50 for cell slides; Beyotime) was applied for 60 min at RT. The nucleus was stained with DAPI (Biosharp, Beijing, China). The images were captured by a fluorescence microscope (Olympus).
5
NF-κB Signaling Pathway Activation
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Cells at a concentration of 2 × 105 cells/mL in 2 mL of medium were plated in a 6-well plate with a 0.17 mm coverslip. Cells were pre-treated with AGS 50 μg/mL for 1 h. To activate the NF-κB signaling pathway, LPS (1 μg/mL) was added to cells and incubated for 30 min. Cells were fixed in 4% paraformaldehyde, and the cell membrane was destroyed by 0.5% Triton X-100 to allow antibodies to enter the nucleus. Antibodies were then blocked with 5% BSA containing 0.1% Triton X-100. Cells were incubated overnight at 4 °C with an NF-κB p65 primary antibody (Santa Cruz Biotechnology, Delaware Ave, Dallas, CA, USA) and 1% BSA (1:200). Cells were incubated with a secondary antibody (Santa Cruz Biotechnology, Delaware Ave, Dallas, CA, USA) and 1% BSA (1:1000) at room temperature in the dark for 2 h. Finally, cell nuclear staining and mounting were conducted using histology mounting medium with DAPI (Sigma-Aldrich, St. Louis, MO, USA).
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