Immunohistochemical staining was performed with sections that had been deparaffinized and rehydrated. Slides were incubated with 0.3% hydrogen peroxide for 20 min before incubation with serum to reduce nonspecific binding. The slides were then incubated overnight with anti-CRP 1 : 200 (Abcam, USA), anti-IL-1β 1 : 100 (Bioss, China), anti-IL-10 1 : 150 (Bioss, China), anti-TNF-α 1 : 100 (Bioss, China), anti-CD68 1 : 100 (Abcam, USA), anti-inducible nitric oxide synthase (iNOS) 1 : 100 (Affinity, USA), and antireceptor activator of NF-κB (RANKL) 1 : 100 (Bioss, China). Then, the slides were washed and incubated with goat anti-mouse secondary antibodies for 30 min (Zhongshan Biotechnology, China), visualized with 3,3-diaminobenzidine tetrahydrochloride (DAB) substrate (Zhongshan Biotechnology, China), and counterstained with haematoxylin. Image-Pro Plus (6.0, Media Cybernetics, Rockville, MD) was used for the semiquantitative analysis, and protein expression was evaluated based on the integrated optical density (IOD).
Anti tnf α
Manufactured by Bioss Antibodies
Sourced in China, United States
Anti-TNF-α is a laboratory reagent used to detect and quantify the presence of Tumor Necrosis Factor alpha (TNF-α) in biological samples. TNF-α is a pro-inflammatory cytokine that plays a crucial role in various physiological and pathological processes. This product provides a specific and sensitive tool for researchers to investigate the role of TNF-α in their studies.
Automatically generated - may contain errors
Lab products found in correlation
Anti il 1β, by Bioss Antibodies (6 mentions)
Anti il 10, by Bioss Antibodies (4 mentions)
Anti il 4, by Bioss Antibodies (3 mentions)
Anti il 6, by Bioss Antibodies (2 mentions)
Rabbit anti inos, by Bioss Antibodies (2 mentions)
Anti arg1, by Bioss Antibodies (2 mentions)
Alexa fluor 488 conjugated goat anti rabbit igg, by Bioss Antibodies (2 mentions)
7266 fluorescence microscope, by Leica (2 mentions)
Anti opn, by Abcam (2 mentions)
Anti crp, by Abcam (1 mentions)
Anti inducible nitric oxide synthase, by Affinity Biosciences (1 mentions)
Rankl, by Bioss Antibodies (1 mentions)
Anti cd68, by Abcam (1 mentions)
Image pro plus 6, by Media Cybernetics (1 mentions)
Goat anti mouse secondary antibodies, by Zhongshan Biotechnology (1 mentions)
Anti cd86, by Bioss Antibodies (1 mentions)
Anti cd8, by Wuhan Servicebio Technology (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (1 mentions)
Horseradish peroxidase conjugated anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Quantity one version 1, by Bio-Rad (1 mentions)
10 protocols using anti tnf α
1
Immunohistochemical Analysis of Inflammatory Markers
2
Immunofluorescence Analysis of Tumor Microenvironment
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Tumor or brain tissues were harvested, fixed in paraformaldehyde, embedded within paraffin and sectioned. Tumor sections (10 μm) were deparaffinized and rehydrated. Sections were boiled in 10 mM citrate buffer (pH 6.0) at 95 °C for 15 min for antigen retrieval. Sections then were blocked with 5% goat serum in PBS for 1 h and incubated with anti-CD8 (1: 800) (Servicebio, China), anti-CD49b (1: 400) (Bioss, China), anti-CD86 (1: 500) (Bioss, China) and anti-TNF-α (1: 500) (Bioss, China) at 4 °C overnight, followed by incubation with Alex 488- or Cy5-conjugated goat-anti-rabbit IgG (1: 500) (Servicebio, China) at room temperature for 1 h. Cell nuclei were labeled with 4′,6-diamidino-2-phenylindole (DAPI). Images were captured using LSCM.
Optical imaging data were analyzed using ImageJ Fiji software32. Total cell numbers were calculated from manual counts of DAPI-labeled nuclei; counts were performed with the Fiji cell counter tool.
Optical imaging data were analyzed using ImageJ Fiji software32. Total cell numbers were calculated from manual counts of DAPI-labeled nuclei; counts were performed with the Fiji cell counter tool.
3
Immunohistochemical Analysis of Neuroinflammation
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The rats were killed by intraperitoneal injection (10% chloral hydrated, 1 ml/100 g) and 4% paraformaldehyde (PFA) was used to fix via cardiac perfusion. Coronal brain slices (15 μm thick) were prepared and used for staining. Slices were washed in PBS for 5 min, three times, then blotted in 5% goat serum for 1 h at room temperature. Slices were incubated with the primary antibodies overnight at 4°C. Primary antibodies used: mouse anti-Iba1 (1:200 Sigma American), mouse anti-GFAP (1:200 Sigma American), rabbit anti-iNOS, anti-Arg1, anti-IL-1β, anti-TNF-α, anti-IL-4, anti-IL-6, and anti-IL-10 (1:200; Bioss, Beijing, China). After having been washed in PBS three times, these slices were incubated with secondary antibodies at 37°C for 2 h. Secondary antibodies: Alexa Fluor 488-conjugated goat anti-rabbit IgG (1:200; Bioss, Beijing, China), Cy3-conjugated goat ant-mouse IgG (1:200; Bioss, Beijing, China). Then these slices were washed three times again and stained with DAPI (Biyuntian, China) for 5 min. Results were imaged using a 7266-fluorescence microscope (Leica, Japan).
4
Quantifying Cytokine Protein Levels
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Cytoplasmic proteins were isolated from treated cells. After determining the protein concentration using the bicinchoninic acid method (Song et al., 2019), proteins were separated using 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After transferring onto a polyvinylidene difluoride membrane, nonspecific staining was blocked with 5% non-fat milk. The membrane was incubated with rabbit polyclonal anti-IL-1β (1:500; Cat# ab9722; Abcam, Cambridge, MA, USA), rabbit polyclonal anti-TNF-α (1:500; Cat# bs-2018R; Bioss, Wuhan, China), and mouse monoclonal anti-IL-6 (1:500, Cat# ab9324; Abcam) at 4°C overnight. The membranes were then probed with horseradish peroxidase-conjugated anti-mouse IgG (1:1000; Cat# 31430; Thermo Fisher Scientific, Inc.) or horseradish peroxidase-conjugated anti-rabbit IgG (1:1000; Cat# 31460; Thermo Fisher Scientific, Inc.) at room temperature for 2 hours. The membranes were visualized using a gel imaging system (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Densitometry was performed using Quantity One version 1.4.6 (Bio-Rad Laboratories, Inc.).
5
Immunofluorescence Assay for Microglial Markers
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Cells were fixed with 4% paraformaldehyde for 10 min and then washed in phosphate-buffered saline (PBS). After being blocked with 5% bovine serum albumin containing 0.5% Triton X-100 for 1 h at 37°C, the microglia were incubated with the following primary antibodies at 4°C overnight: mouse anti-Iba1 (1:200, Bioss, Beijing, China), rabbit anti-iNOS, anti-Arg1, anti-IL-1β, anti-TNF-α, anti-IL-4, anti-IL-6 and anti-IL-10 (1:200, Bioss). After being washed in PBS three times, the cells were incubated with the following secondary antibodies at 37°C for 1 h: Alexa Fluor 488-conjugated goat anti-rabbit IgG (1:200, Bioss) or Alexa Fluor Cy3-conjugated goat anti-mouse IgG (1:200, Bioss). After being washed in PBS three times, the cells were incubated with 4′,6-diamidino-2-phenylindole (DAPI; Biyuntian, China) for 5 min. The cells were then washed in PBS and observed under a 7266-fluorescence microscope (Leica, Japan).
6
Western Blot Analysis of Liver Proteins
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Proteins were obtained from liver tissues and liver cells using radioimmunoprecipitation assay (RIPA) buffer (Beyotime, Shanghai, China), while protein concentration was assayed using a bicinchoninic acid (BCA) protein assay kit (Beyotime). Proteins were separated using sodium salt-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes. After blocking, membranes were incubated overnight with primary antibodies at 4°C (anti-TNF-α [1:1000; Bioss]; anti-OPN [1:1000; Abcam]; anti-CYP7A1 antibodies [1:1000; Invitrogen]; anti-β-actin [1:2000; Abcam]). Horseradish peroxidase (HRP)-conjugated goat anti-rabbit (1:10,000; Jackson, PA, USA) was used as the secondary antibody. Finally, membranes were visualized using enhanced chemiluminescence (Beyotime) and analyzed on Image J 1.8.0 (National Institutes of Health, Bethesda, MD, USA).
7
Immunohistochemical Analysis of Inflammatory Markers
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Sections were stained with the following primary antibodies for immunohistochemical analyses: anti-TNF-α (dilution ratio: 1:400; Bioss Inc., Woburn, MA, USA), anti-IL-1β (1:400; Bioss Inc.), anti-TGF-β1 (1:400; Bioss Inc.), anti-OPN (1:400; Abcam, Cambridge, UK), and anti-CTSK (1:400; Abcam). Briefly, after deparaffinization and rehydration, the sections were incubated with 3% hydrogen peroxide (Abcam) to quench the endogenous peroxidase activity; thereafter, the sections were blocked with specific animal serum for nonspecific binding and then incubated with specific primary antibodies and VECTASTAIN Elite ABC Rabbit IgG Kit (Vector Laboratories, Inc., Burlingame, CA, USA) with biotinylated secondary antibody and ABC Reagent. Subsequently, 3,3′-diaminobenzidine (Abcam) was applied, and hematoxylin (Fujifilm Wako Pure Chemical Corp.) staining was performed for counterstaining. The levels of TNF-α, IL-1β, TGF-β1, and OPN were semi-quantified by the percentage of immuno-positive stained areas (%).
8
Investigating Gut Inflammation Pathways
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We used the following materials: Con A was acquired from Sigma-Aldrich (St. Louis, MO). BBR, alcian blue stain kit, methylprednisolone (MP), ampicillin, neomycin, metronidazole, and vancomycin were afforded by Solarbio (Beijing, China). Anti-Caspase-3, anti-Bax, and anti-F4/80 antibodies were offered by cell signaling technology (Boston, MA). Anti-TNF-α, anti-interferon-γ, anti-IL-1β, anti-IL-17A, and anti-IL-10 antibodies were purchased from Bioss (Beijing, China). Rabbit anti-mouse primary antibodies, including zonula occludens-1 (ZO-1), occludin, TLR4, NF-κB P65, inhibitor of NF-κB (IκB), phospho-inhibitor of NF-κB (p-IκB), and β-actin, were obtained from Abcam (Cambridge, UK). Allophycocyanin-conjugated anti-CD4, fluorescein isothiocyanate–conjugated anti-IL-17A, and phycoerythrin-conjugated anti-Foxp3 antibodies were purchased from BD (Franklin Lakes, NJ). LPS ELISA kits were supplied by Cloud-clone (Hubei, China).
9
Western Blot Analysis of Ischemic Injury
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At different reperfusion times, protein was extracted from the infarcted hemispheres and cultured cells by a protein extraction kit (Beyotime Biotech) as described in our previous report (Li et al., 2015 (link)). Protein samples were next electrophoresed and transferred to polyvinylidene difluoride filter (PVDF) membranes. Then block the membrane with 5% fat-free milk at room temperature for 2 h, and incubate with primary antibodies as follows: anti-Tubulin (Cell Signaling), anti-Cx43 (Cell Signaling), anti-TLR4 (Proteintech), anti-MyD88 (Cell Signaling), anti-NF-κB p65 (Proteintech), anti-IL-1β (Bioss), and anti-TNF-α (Bioss) at 4°C overnight. Next day, incubate with secondary horse anti-mouse or goat anti-rabbit antibodies conjugated with horseradish peroxidase (Cell Signaling) for 2 h and visualize by an enhanced chemiluminescence system (ECL). Relative protein levels were quantified after normalization to Tubulin.
10
Gestational Immune Profile Analysis
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Total protein was extracted from spleen tissues isolated from five pregnant CBA/J mice from each group on day 14 of gestation using RIPA lysis buffer (Beyotime Institute of Biotechnology). Protein (30 µg/lane) was quantified using a bicinchoninic acid assay kit (Beyotime Institute of Biotechnology) and separated by 10% SDS-PAGE and transferred to PVDF membranes. The membranes were blocked with 10% skim milk at room temperature for 4 h. Subsequently, the membranes were incubated at 4°C overnight with the following primary antibodies: Anti-FoxP3 (cat. no. bs-0269R; 1:1,000; BIOSS), anti-TNF-α (cat. no. A0277; 1:1,000; ABclonal Biotech Co., Ltd.), anti-IFN-γ (cat. no. bs-0480R; 1:1,000; BIOSS), anti-IL-4 (bs-0581R; 1:1,000; BIOSS) and anti-β-actin (cat. no. AC026; 1:2,000; ABclonal Biotech Co., Ltd.). Subsequently, the membranes were incubated with a goat anti-rabbit IgG horseradish peroxidase-conjugated secondary antibody (1:2,000; cat. no. bs-0295G-HRP; BIOSS). Protein bands were visualized using ECL Plus Western Blotting Detection reagents (EMD Millipore). Blots were performed in triplicate and protein expression was quantified using ImageJ 2× software (National Institutes of Health) with β-actin as the loading control.
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