The TNF-α and COX-2 levels in liver homogenate was performed using commercial ELISA kit obtained from Sigma Aldrich Chemical Co. according to the manufacturer's instructions. The quantities of TNF-α and COX-2 were expressed as ng/mg protein. The protein was calculated using bovine serum albumin as standard [61 (link)]. Furthermore, NF-κBp65 subunit expression was assessed immunohistochemically in formalin-fixed paraffin-embedded rat liver using polyclonal antibody (PA5-16545, ThermoFisher).
Pa5 16545
Manufactured by Thermo Fisher Scientific
Sourced in United States
The PA5-16545 is a laboratory equipment product from Thermo Fisher Scientific. It is designed for use in scientific research and analysis applications. The core function of this product is to provide a specific set of capabilities to support laboratory workflows, but a detailed description cannot be provided while maintaining an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Fitc conjugated igg, by Jackson ImmunoResearch (1 mentions)
Alexa fluor 555 conjugated igg, by Thermo Fisher Scientific (1 mentions)
Sc 53564, by Santa Cruz Biotechnology (1 mentions)
Abt123, by Merck Group (1 mentions)
Av44268, by Merck Group (1 mentions)
G9545, by Santa Cruz Biotechnology (1 mentions)
A2547, by Thermo Fisher Scientific (1 mentions)
P jnk, by Cell Signaling Technology (1 mentions)
Af1935, by R&D Systems (1 mentions)
Chip kit one step, by Abcam (1 mentions)
Hmc3 human microglial cell line, by American Type Culture Collection (1 mentions)
Anti p 1 κbα, by Thermo Fisher Scientific (1 mentions)
Dapi h 1200, by Vector Laboratories (1 mentions)
6 protocols using pa5 16545
1
TNF-α and COX-2 Quantification in Liver
2
Immunofluorescent Localization of NF-κB and COX-2
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We used the immunofluorescent method as outlined in our previous study (Lee et al., 2019 (link)) to confirm the location of NF-κB in the nucleus or the cytoplasm, and the expression level of COX-2 in the cytoplasm. Paraffin-lung tissue samples were placed on slides and were incubated at room temperature for 1 hr with primary antibodies including NF-κB (PA5-16545, Thermo Fisher Scientific) and COX-2 (PA1-9032, Invitrogen, Carlsbad, CA, USA). Secondary antibodies such as FITC-conjugated IgG (315-095-003, Jackson Immunoresearch, West Grove, PA, USA) or Alexa Fluor 555-conjugated IgG (A-21127, Thermo Fisher Scientific) were used to counterstain and were used by DAPI (62249, Thermo Fisher Scientific). All images were acquired using a K1-Fluo confocal microscope (Nanoscope System, Daejeon, Korea) and the fluorescent intensity was analyzed by the software in the confocal microscope.
3
Protein Expression Analysis of Fibrosis Markers
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From each AAA sample 25 μg of denatured protein was separated on 4–12% polyacrylamide by electrophoresis and then transferred to PVDF membrane. The membranes were then incubated with collagen type-1 (Millipore #ABT123, 1:2000), TGF-β1 (Sigma #AV44268, 1:1000), α-SMA (Sigma #A2547, 1:1000) and KFkB (Thermo-Fisher #PA516545). GAPDH (Sigma # G9545, 0.1 mg/ml) and Caveolin-1 (Santa Cruz # sc-53564, 1:1000) were used as loading controls. Membranes were treated with HRP-conjugated secondary antibody at room temperature for 1 h and evaluated with chemiluminescence. Blot band intensities were analyzed using ImageJ software.
4
Immunohistochemical Analysis of Intestinal Apoptosis
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Sections at 5 µm incubated at 56 °C overnight were dewaxed and rehydrated. Sections after antigen retrieval and blocking were incubated in rabbit-polyclonal primary antibodies caspase-12 (1/2000, NBP1-77290, Novus Biologicals, Missouri, USA), GRP78/BİP (1/3000, LS-C312961, Lifespan Biosciences, Washington, USA), NF-κB/p65 (1/100, PA5-16545, Thermo Fisher Scientific, Massachusetts, USA) and p-JNK (1/50, 9251, Cell Signaling Technology, Massachusetts, USA) as described before.27 (link) AEC chromogen (AEC, ab64252, Abcam, Cambridge, UK), was applied. Sections were counterstained with hematoxylin.
The immunoreactivities of GRP78, NF-κB, caspase 12, and p-JNK in the intestinal tissue were evaluated in five fields/sections under a light microscope by blinded observation at 200x magnification. For each section, an HSCORE value was calculated by multiplying the percentage of immune-positive cells and the intensity of staining and then summing the values.29 (link)
The immunoreactivities of GRP78, NF-κB, caspase 12, and p-JNK in the intestinal tissue were evaluated in five fields/sections under a light microscope by blinded observation at 200x magnification. For each section, an HSCORE value was calculated by multiplying the percentage of immune-positive cells and the intensity of staining and then summing the values.29 (link)
5
Investigating Transcriptional Regulation of LOX-1
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We identified the binding sites for NF-kB, Oct-1 and HIF-1α in the promoter region of the human OLR1 gene from the UCSC Genome Browser (http://genome.ucsc.edu ). To identify whether the transcription factors NF-κB and HIF-1α control the expression of LOX-1 in microglia, we performed a chromatin immunoprecipitation assay using the ChIP Kit-One Step (ab117138; Abcam, Cambridge, UK) with the HMC3 human microglial cell line (CRL-3304; American Type Culture Collection, Manassas, VA). According to the manufacturer’s instructions, we collected OGD-treated microglial cells, fixed them with 1% formaldehyde and fragmented them with sonication. We used antibodies against NF-κB (PA5-16545; Thermo Fisher Scientific) and HIF-1α (AF1935; R&D Systems) and primer sets for NF-κB and HIF-1α (Additional file 3 : Table S2).
6
Immunostaining of γH2AX, p53, and NF-kB
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Immunostaining of γH2AX, p53, and NF-kB was followed by instructed protocol. In brief, cultured glomerular endothelial cells were fixed in 4% PFA for 15 minutes, permeabilized with 0.5% Triton X-100 in PBS for 15 minutes, and blocked with 3% bovine serum albumin in PBS for 60 minutes. All the procedures were performed at room temperature. Cells were then incubated with primary antibody, anti-γH2AX (1:500, rabbit monoclonal, 9718, Cell Signaling Technology, MA, USA), anti-p53 (1:50, rabbit polyclonal, 10442-1-AP, Proteintech, IL, USA), anti-NF-kB (1:100, rabbit polyclonal, PA5-16545, Thermo Fisher, IL, USA), and anti-p-IκBα (1:50, mouse monoclonal, santa cruz, sc-8404, TX, USA) at 4 °C overnight. Cells were incubated with Alexa Fluor Plus 488 goat anti-rabbit IgG secondary antibody (1:500, A32731, Thermo Fisher Scientific, Tokyo, Japan), at room temperature for 1 hour. Nuclei were counterstained with DAPI (H-1200, Vector Laboratories, CA, USA).
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