Male Sprague-Dawley (SD) rats, 6 weeks old (200–230 g), were provided by the experimental animal center of Binzhou Medical University. The experimental animals were treated according to the guidelines approved by university committee on the use and care of laboratory animals. Trizol and the two-step PCR detection kits were purchased from the TaKaRa Co. (Dalian, China). Anti-α-SMA antibodies, anti-TGF-β1 antibody, anti-MMP-1 antibody, and anti-NF-κB p65 antibody for immunocytochemistry were purchased from the Boster Co. (Wuhan, China). The light microscope and camera were purchased from Nikon Corporation (Japan). All other chemicals and reagents were of analytical grade.
Anti nf κb p65 antibody
Manufactured by Boster Bio
Sourced in China
The Anti-NF-κB p65 antibody is a research-use laboratory reagent that targets the p65 subunit of the NF-κB transcription factor. It is designed for use in various techniques, such as Western blotting, immunohistochemistry, and immunocytochemistry, to detect and analyze the expression and localization of the p65 protein.
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Lab products found in correlation
Anti tgf β1 antibody, by Boster Bio (1 mentions)
Trizol, by Takara Bio (1 mentions)
Goat serum, by Boster Bio (1 mentions)
Cy3 labeled goat anti rabbit igg h l, by Beyotime (1 mentions)
Triton x 100, by Beyotime (1 mentions)
Anti il 6 antibody, by Boster Bio (1 mentions)
Tnf α antibody, by Affinity Biosciences (1 mentions)
3 protocols using anti nf κb p65 antibody
1
Sprague-Dawley Rat Fibrosis Model
2
Immunofluorescence Analysis of NF-κB p65
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Cells grown on a slide were fixed with 4% paraformaldehyde for 30 min and then permeabilized with 0.5% Triton X-100 (Beyotime Biotechnology, China) for 15 min at room temperature. After incubating at room temperature for 30 min with goat serum (Boster Biotechnology, China), anti-NF-κB p65 antibody (cat. no. ab16502, Abcam) was added (2.5:1000) and incubated overnight at 4°C. After incubation with Cy3-labeled goat anti-rabbit IgG (H+L) (Beyotime Biotechnology, China) for 1 h, the nuclei were counterstained with DAPI (Beyotime Biotechnology, China) for 10 min. The slides were mounted, and the expression of p65 was observed under a fluorescence microscope (Olympus IX71, Japan).
3
Immunohistochemistry for Inflammatory Markers
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The immunohistochemistry technique was used for the levels of NF-κB, IL-6, TNF-α, and IL-1β expression according to the methodology described previously (23 (link)). Paraffin sections were deparaffinized and underwent antigen repairing. Then, sections were incubated with the primary antibody (1:50) at room temperature for 1-2 hours. Subsequently, sections were incubated with the secondary antibody. Then, they were visualized by incubating them with 3, 3'-diaminobenzidine etrahydrochloride (DAB) (Servicebio, China). The primary antibodies were the following: IL-1β polyclonal antibody (Bioworld Technology, co. Ltd., China; Cat.no. BS6067; rabbit), anti-IL-6 antibody (Boster, China; Cat. no. BA4339; rabbit IgG), TNF-α antibody (Affinity Biosciences, China; Cat. no. AF7014; rabbit polyclonal), and anti-NF-κBp65 antibody (Boster, China; Cat. no. BA0610; rabbit IgG).
The sections were evaluated with a high-power objective lens (25×) using optical microscopy (E100 LED, Nikon Corporation, Japan). The immunostaining intensity of pro-inflammation was evaluated by two examiners under double-blinded conditions. Possible scores were as follows. Score 1: 0% of positive cells. Score 2: <10% of positive cells or isolated cells. Score 3: 11–50% of positive cells. Score 4: >50% of positive cells (25 (link)).
The sections were evaluated with a high-power objective lens (25×) using optical microscopy (E100 LED, Nikon Corporation, Japan). The immunostaining intensity of pro-inflammation was evaluated by two examiners under double-blinded conditions. Possible scores were as follows. Score 1: 0% of positive cells. Score 2: <10% of positive cells or isolated cells. Score 3: 11–50% of positive cells. Score 4: >50% of positive cells (25 (link)).
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