Immunohistochemistry staining were performed according previous studies [14 (link),28 (link)]. Livers were paraffin-embedded and cut into 5 μM-thick sections. After that, these sections were dewaxed and rehydrated, followed by heat-induced epitope retrieval. Subsequently, sections were placed into hydrogen peroxide solution in order to suppress endogenous peroxidase activity. Then the sections were washed with PBS for three times and blocked by BSA solution. Immunohistochemical staining was conducted by incubating primary antibodies for 24 h overnight at 4 °C and then secondary antibodies for 1 h at room temperature. The primary antibodies used for EC toxicity animal experiments were shown as follows: Anti-TNFα (Abways, CY5992, 1:200), Anti-IL-1β (Servicebio, GB11113, 1:200), Anti-NLRP3 (Abways, CY5651, 1:200), Anti-4-HNE (Bioss, bs-10802R, 1:300). The primary antibodies used for Fer-1 and tBHQ rescue experiments were shown as follows: TNFa (Proteintech, 26405-1-AP, 1:200), NLRP3 (Proteintech, 19771-1-AP, 1:200), IL-1β (Servicebio, GB11113, 1:800), 4-HNE (Bioss, bs-6313R, 1:200). Sections were stained with freshly prepared DAB and hematoxylin, then dehydrated and sealed with neutral gum. Images were detected using a Nikon microscope.
Il 1β
Manufactured by Wuhan Servicebio Technology
Sourced in China
IL-1β is a cytokine that plays a key role in the inflammatory response. It is involved in the regulation of immune responses, hematopoiesis, and inflammation. IL-1β is produced by activated macrophages and is a primary mediator of the body's response to injury, infection, and antigenic challenge.
Automatically generated - may contain errors
Lab products found in correlation
Tnf α, by Wuhan Servicebio Technology (6 mentions)
Nlrp3, by Wuhan Servicebio Technology (4 mentions)
Interleukin 6 (il 6), by Wuhan Servicebio Technology (3 mentions)
Caspase 1, by Wuhan Servicebio Technology (3 mentions)
Goat anti rabbit igg, by Wuhan Servicebio Technology (2 mentions)
Goat anti mouse igg, by Wuhan Servicebio Technology (2 mentions)
Il 18, by Wuhan Servicebio Technology (2 mentions)
Image pro plus 6, by Wuhan Servicebio Technology (2 mentions)
4 hne, by Bioss Antibodies (1 mentions)
Bs 6313r, by Bioss Antibodies (1 mentions)
Anti il 1β, by Wuhan Servicebio Technology (1 mentions)
Bs 10802r, by Bioss Antibodies (1 mentions)
Anti 4 hne, by Bioss Antibodies (1 mentions)
Neutral balsam, by Wuhan Servicebio Technology (1 mentions)
Bsa solution, by Wuhan Servicebio Technology (1 mentions)
Dab chromogenic kit, by Wuhan Servicebio Technology (1 mentions)
Fv3000, by Olympus (1 mentions)
Nephrin, by Thermo Fisher Scientific (1 mentions)
Tunel kit, by Wuhan Servicebio Technology (1 mentions)
Podocin, by Proteintech (1 mentions)
17 protocols using il 1β
1
Immunohistochemical Analysis of Liver Inflammation
2
Immunohistochemical Analysis of Inflammatory Cytokines
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Wuzhou Liupao tea was sourced from Wuzhou tea factory (Zhuang Autonomous Region, Guangxi, China). The Huoxiang Zhengqi oral liquid was purchased from MINJI Pharmaceutical Co., Ltd (Jiangxi, China). Lard oil was bought from GLOD GIFT FOOD Co., Ltd (Zhejiang, China). EDTA (pH 9.0) antigen repair solution, PBS buffer (pH 7.4), BSA solution, neutral balsam, TNF-α, IL-6, IL-1β, goat anti-rabbit IgG, goat anti-mouse IgG, and the DAB chromogenic kit were purchased from Service-Bio Co., Ltd (Wuhan, China). Ethanol and xylene were acquired from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China).
3
Immunofluorescence Analysis of Kidney Injury
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TUNEL kits (Servicebio, G1501) were used to detect apoptotic cells in paraformaldehyde-fixed kidney tissues, and the appropriate experimental protocols were applied. For immunofluorescence in tissues, paraffin sections were blocked with 10% donkey serum and incubated overnight at 4 °C with primary antibodies nephrin (1:100, Invitrogen, PA5-106921), podocin(1:100, Proteintech, 20384-1-AP), cd2ap(1:100, Invitrogen, PA5-51894), TNF-α(1:500, Servicebio, GB11188), IL-6(1:500, Servicebio, GB11117), IL-1β(1:300, Servicebio, GB11113), and LC3(1:500, Proteintech, 14600-1-AP), followed by secondary antibodies. Finally, the nuclei were restained with DAPI and observed under a confocal microscope (Olympus, FV3000).
For immunofluorescence in cells, each group of cells were given the corresponding treatment and sequentially subjected to cell rupture, serum closure, incubated with the corresponding primary antibodies nephrin (1:100, Invitrogen, PA5-106921), podocin (1:100, Proteintech, 20384-1-AP), cd2ap (1:100, Invitrogen, PA5-51894), and LC3(1:500, Proteintech, 14600-1-AP) overnight at 4 °C and secondary antibodies. Finally, after DAPI restaining nuclei, the cells were observed under confocal microscopy.
For immunofluorescence in cells, each group of cells were given the corresponding treatment and sequentially subjected to cell rupture, serum closure, incubated with the corresponding primary antibodies nephrin (1:100, Invitrogen, PA5-106921), podocin (1:100, Proteintech, 20384-1-AP), cd2ap (1:100, Invitrogen, PA5-51894), and LC3(1:500, Proteintech, 14600-1-AP) overnight at 4 °C and secondary antibodies. Finally, after DAPI restaining nuclei, the cells were observed under confocal microscopy.
4
Immunohistochemical Analysis of Inflammatory Cytokines in Spinal Tissues
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Isolated spines and adjacent vertebral bodies were fixed in 4% paraformaldehyde, decalcified, dehydrated, cleared with dimethylbenzene and embedded in paraffin (Tissue Tek processor and Leica embedder, Buffalo Grove, IL). The sections (5 μm) were incubated with 3% hydrogen peroxide to block endogenous peroxidase activity for 10 min and 5% bovine serum albumin was used to block nonspecific binding sites for 30 min at 37°C. Following incubation with the primary antibody (IL-1β 1:1,000, IL-6 1:800, TNF-α 1:1,000, Servicebio, Wuhan, China) overnight at 4°C, the sections were treated with diaminobenzidine-based peroxidase substrate (DAB Horseradish Peroxidase Color Development Kit, Beyotime Biotechnology, Shanghai, China) to visualize the immunoreactivity. The images were acquired under 50× and 400× magnification using the light microscopy mode of the fluorescence microscope and the intensity was quantified by the Image-Pro Plus 6.0 software.
5
Immunohistochemical Analysis of Inflammatory Markers
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Hematoxylin and eosin (H&E) staining and immunohistochemical (IHC) staining were performed according to the standard protocols by Servicebio (Wuhan, China). IHC staining was conducted with primary antibodies of HIF-1α (Abcam, Cambridge, UK), CD31 (Abcam), IL-1β (Servicebio), NLRP3 (Servicebio) and Caspase-1 (Servicebio) at 4°C overnight, then incubated with HRP-labeled Goat Anti-Rabbit IgG (H+L) (Servicebio) for 50 min at room temperature. Diaminobenzidine (DAB) was used to visualize the antigen-antibody reaction. The slides were photographed by microscopy (Olympus BX53). The average integrated optical density (IOD) of HIF-1α, CD31, IL-1β, NLRP3 and Caspase-1 in three or five randomly selected areas for each group were calculated using Image-Pro Plus 6.0 (Media Cybernetics, Inc.).
6
Sciatic Nerve Adhesion Immunohistochemical Analysis
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Immunohistochemistry was performed to assess the expression of HSP72, CD68 (activated macrophages marker), CD163 (marker for M2 macrophages), tumor necrosis factor α (TNF-α) and interleukin 1β (IL-1β) (n = 3 per group) at two weeks after surgery and the expression of HSP 72 and α-SMA (marker for myofibroblasts) at six weeks after surgery.
The sciatic nerves were harvested from the zone of adhesion. After formalin-fixed and paraffin-embedded, the specimens were cut longitudinally into tissue sections (3 μm in thickness). After quenching of endogenous enzymes and antigen retrieval, the tissue sections were incubated with the antibodies against HSP72 (1:100; Enzo life Sciences), CD68 (1:100, Servicebio, China), CD163 (1:100, Servicebio, China), TNF-α (1:100, Servicebio, China), IL-1β (1:100, Servicebio, China), and α-SMA (1: 400; Cell Signaling Technology) at 4 °C overnight. Then horseradish peroxidase-conjugated goat anti-rabbit antibodies were utilized as secondary antibodies. 3, 3-diaminobenzidine tetrahydrochloride (DAB; Zhongshan Golden Bridge Biotechnology, China) peroxidase substrate solution was used to visualize staining. The images were observed and photographed under a microscope, and high-power fields (200×) were chosen and captured. Quantitative analysis is the average optical density analysis after dyeing by ImageJ software.
The sciatic nerves were harvested from the zone of adhesion. After formalin-fixed and paraffin-embedded, the specimens were cut longitudinally into tissue sections (3 μm in thickness). After quenching of endogenous enzymes and antigen retrieval, the tissue sections were incubated with the antibodies against HSP72 (1:100; Enzo life Sciences), CD68 (1:100, Servicebio, China), CD163 (1:100, Servicebio, China), TNF-α (1:100, Servicebio, China), IL-1β (1:100, Servicebio, China), and α-SMA (1: 400; Cell Signaling Technology) at 4 °C overnight. Then horseradish peroxidase-conjugated goat anti-rabbit antibodies were utilized as secondary antibodies. 3, 3-diaminobenzidine tetrahydrochloride (DAB; Zhongshan Golden Bridge Biotechnology, China) peroxidase substrate solution was used to visualize staining. The images were observed and photographed under a microscope, and high-power fields (200×) were chosen and captured. Quantitative analysis is the average optical density analysis after dyeing by ImageJ software.
7
Liver Protein Analysis by Western Blot
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The liver tissue was lysed in a RIPA buffer (Servicebio, China) that was supplemented with proteinase (Servicebio, China), phosphatase inhibitors (Servicebio, China), and PMSF (Servicebio, China). The lysates were then centrifuged at 12,000 rpm at 4 °C for minutes to remove debris, and the resulting supernatants were collected for electrophoresis. Western blotting was performed according to the protocol described in our previous study [26 (link)]. The primary antibodies used for the western blotting were TNF-α (Servicebio, China), IL-1β (Servicebio, China), and Tubulin-β (Abways Technology, Shanghai, China). Quantitative analysis of the western blotting was performed using ImageJ software.
8
Immunofluorescence Staining of Brain Tissue
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Brain tissue samples were washed with PBS three times and sealed with 5% BSA blocking solution at room temperature for 1 h. The corresponding primary antibody was selected to incubate the sectioned tissue, and the antibody information is as follows: LC3 (GB13431, Servicebio, China), Lamp1 (bs-1970R, Bioss, China), NLRP3 (GB11300, Servicebio, China), Caspase-1 (GB11383, Servicebio, China), IL-1β (GB11113, Servicebio, China), IBA-1 (GB12105, Servicebio, China), and iNOS (GB11119, Servicebio, China). The samples were then refrigerated overnight at 4°C. The next day, the slices were cleaned three times, and the secondary antibody Fitc (G1222, Servicebio, China) or Cy3 (GB123, Servicebio, China) was incubated at room temperature in the dark for 1 h. DAPI was used to stain the tissues’ nuclei, and the samples were cleaned and sealed with a cover glass. A fluorescence microscope was used to observe and collect fluorescence images. The fluorescence intensity was analyzed by ImageJ software (Meng et al., 2016 (link)).
9
Liver Expression of Circadian and Inflammatory Genes
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Total RNA was extracted from the livers of dogs using the Trizol reagent (Servicebio, China). Reverse transcription of total RNA was performed using a reverse transcription kit (ThermoFisher Scientific, Waltham, MA, USA). The product was used for subsequent quantitative real-time polymerase chain reaction (qRT-PCR) detection of Bmal1, TNF-α, and IL-1β (Servicebio, China). GAPDH was used as an internal reference for normalization. The primer sequences used for Bmal1 were as follows: (forward) 5′-AAGGGAAGCTCACAGTCAGAT and (reverse) 5′-GGACATTGCGTTGCATGTTGG. The primers used for TNF-α were as follows: (forward) 5′-CCTGGAGCCAAATTCGAGTG and (reverse) 5′-CGTGTGGGTTCTGTCTCGTG. The primers used for IL-1β were as follows: (forward) 5′-CCAGCTCAGAGGCTCTTGTG and (reverse) 5′-AGGGCCAGGTCCTGAGAAAT.
10
Immunofluorescence Staining of Tissue Samples
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The samples were washed with PBS, fixed with 4 % paraformaldehyde for 30 min, according to experimental requirements, were incubated with anti-Pdpn (proteintech, 1:200), anti-Grem1 (CST, 1:50), anti-Cd200 (proteintech, 1:200), anti-Ctsk (Abcam, 1:200), anti-Ptgs2 (proteintech, 1:200), anti-vinculin (proteintech, 1:200) or anti-YAP (CST, 1:200) at 4 °C for 8–12 h. After that, samples incubated with goat anti-rabbit IgG (Servicebio, Alexa Fluor 488, 1:300), goat anti-rabbit IgG (Servicebio, Cy3, 1:300) or goat anti-mouse IgG (Servicebio, Alexa Fluor 488, 1:200) for 1 h at room temperature. Nuclear staining was conducted with DAPI (Sigma), and F-actin stained by rhodamine phalloidin (Beyotime). Tissue samples were fixed in 4 % paraformaldehyde. After decalcification, all samples were embedded in paraffin and sectioned at 6-μm slices. The tissue sections were immunofluorescently stained with primary antibodies targeting Sry (Santa Cruz, 1:200), CD11b (Servicebio, 1:500), TNF-α (Uscn Life Science Inc., 1:1000), IL-1β (Servicebio, 1:1200) and IL-10 (Servicebio, 1:1000), and the goat anti-rabbit IgG (Servicebio, Alexa Fluor 488, 1:400) and goat anti-rabbit IgG (Servicebio, Cy3, 1:500). DAPI was used to stain the nuclei. The images were observed by a fluorescence microscope (Olympus CKX53) or Nikon confocal microscope (Nikon).
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