For immunofluorescent staining, non-specific binding was blocked using 10% blocking serum in PBS for 1 hour, and the tissue samples were then incubated with primary antibodies to AhR (ab84833, Abcam), nestin (clone 10C2, Abcam), and GFP (clone 4B10, Cell Signaling Technology Inc.) overnight at 4°C. Secondary antibodies conjugated with fluorescence were added, and slides were incubated at room temperature for 1 h. Isotype-matched negative control antibodies (R&D Systems) were used under the same conditions. Nuclei were counterstained with 6-diamidino-2-phenylindole, dihydrochloride (DAPI) (Sigma). The sections were mounted with the ProLong Gold Anti-fade Kit (Molecular Probes, Grand Island, NY) and observed by a NIKON ECLIPSE Ti-U microscope equipped with DS-Fi2 camera (NIKON, USA).
Ab84833
Manufactured by Abcam
Sourced in United Kingdom
Ab84833 is a laboratory equipment product. It is a core component used for specific experimental procedures. The detailed specifications and intended use are not available for this product.
Automatically generated - may contain errors
Lab products found in correlation
Ds fi2 camera, by Nikon (2 mentions)
Prolong gold anti fade kit, by Thermo Fisher Scientific (2 mentions)
Ab192579, by Abcam (2 mentions)
Eclipse ti u microscope, by Nikon (2 mentions)
Ab52903, by Abcam (1 mentions)
Ab179695, by Abcam (1 mentions)
Alexa fluor dye, by Thermo Fisher Scientific (1 mentions)
Ep568y, by Abcam (1 mentions)
Erk 4695, by Cell Signaling Technology (1 mentions)
Ab181602, by Abcam (1 mentions)
Gsh px, by Nanjing Jiancheng (1 mentions)
Ab104139, by Abcam (1 mentions)
Lsm710 imaging system, by Zeiss (1 mentions)
Ab9535, by Abcam (1 mentions)
Nanozoomer 2.0 rs, by Hamamatsu Photonics (1 mentions)
9 protocols using ab84833
1
Immunofluorescent Staining for AhR, Nestin, and GFP
2
Quantitative Immunofluorescence Analysis of Lung Tissues
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Sectioned lung tissues were first blocked using 5% w/v bovine serum albumin for 1 h, followed by incubation with the primary antibodies against Smad3 (EP568Y, Abcam), phospho-Smad3 (ab52903; Abcam), TGFβ1 (Ab179695; Abcam), RhoA-GTPase (26904; New East Biosciences), AhR (Ab84833; Abcam), and EpCAM (G8.8; ThermoFisher), respectively, overnight at 4°C. The sample sections were then incubated with secondary antibodies conjugated with Alexa Fluor dyes (ThermoFisher) at room temperature for 1 h. Isotype-matched negative control antibodies (R&D Systems) were used under the same conditions. The nuclei were counterstained with 6-diamidino-2-phenylindole, dihydrochloride (Solarbio, Beijing, China). Sections were mounted with the ProLong Gold Anti-fade Kit (Molecular Probes) and observed with a Nikon Eclipse Ti-U microscope equipped with a DS-Fi2 camera (Nikon). To determine the fluorescence signal in tissue sections, fluorescent-positive cells in four different high-power fields (500 μM2) from each slide were quantified using ImageJ v1.50e (National Institutes of Health). At least two tissue sections per mouse were included for analysis and presented as mean fluorescence intensity per square micrometer.
3
Propagation of Recombinant Newcastle Disease Viruses
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Chicken embryonic fibroblasts (DF-1) were purchased from the Cell Resource Center of Shanghai Institutes for Biological Sciences of the Chinese Academy of Sciences. BSR-T7/5 cells stably expressing the T7 phage RNA polymerase were a kind gift from Prof. Zhigao Bu (Harbin Veterinary Research Institute, China). DF-1 and were BSR-T7/5 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and antibiotics and were cultured at 37 °C under 5% CO2. The parental NDV (rSS1GFP) and the mutant NDV harboring NLS mutation in the M protein (rSS1GFP-M/NLSm) were generated in our previous study [22 (link)]. The two viruses were plaque purified three times in DF-1 cells and propagated once in specific-pathogen-free (SPF) embryonated chicken eggs. Primary antibody mouse anti-Tubulin monoclonal antibody (sc-53646), mouse anti-GAPDH monoclonal antibody (sc-66163), mouse anti-Lamin B1 monoclonal antibody (sc-56143), and mouse anti-GFP monoclonal antibody (sc-9996) were purchased from Santa Cruz Biotechnology (USA). Mouse anti-PROX1 monoclonal antibody (ab33219), and rabbit anti-AHR polyclonal antibody (ab84833) were purchased from Abcam (UK).
4
Eurotium cristatum Brick Tea Analysis
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Raw PDT leaves, FBT, and Hua Juan Tea (HJT) were purchased from the Baishaxi Tea Factory Co., Ltd. (Yiyang, Hunan, China). The Eurotium cristatum strains were isolated from Baishaxi brick tea using the direct separation technique. Tea-lyophilized powder was prepared at the Hunan Agricultural University. Cigarettes were purchased from the Hunan China Tobacco Industry Co., Ltd. (Changsha, Hunan, China). Vacuum diaphragm pumps were purchased from Kamoer, KVP15-KL-1 (Shanghai, China). Analytical kits for GSH-Px, SOD, and MDA were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, Jiangsu, China). ELISA kits for the analysis of tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), IL-8, and IL-1β were purchased from Wuhan Hualianke Biotechnology Co. Ltd. (Wuhan, Hubei, China). Antibodies against p-p38 (4511S), p38 (8690S), p-Jun N-terminal kinase (p-JNK, (9251S)), JNK (9252S), p-extracellular-regulated kinase (p-ERK, (4370S)), and ERK (4695S) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Monoclonal antibodies against PXR (ab192579), AhR (ab84833), and GAPDH (ab181602) were purchased from Abcam (Cambridge, UK). All other chemicals and reagents were at analytical grade.
5
Analytical Methods for Oxidative Stress
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Analytical kits for SOD, GSH-Px and MDA were purchased from the Nanjing Jiancheng Bioengineering Institute (Nanjing, China). ELISA kits for the analysis of tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), IL-8, and IL-1β were purchased from Wuhan Hualianke Biotechnology Co. Ltd. (Wuhan, China). Extracellular signal-regulated kinases (ERK), phospho-ERK (p-ERK), c-Jun N-terminal kinase (JNK), phosphor-JNK (p-JNK), p38 and phosphor-p38 (p-p38) were purchased from Cell Signaling Technology (Danvers, MA, USA). Monoclonal antibodies against PXR (ab192579), AhR (ab84833), and GAPDH (ab181602) were purchased from Abcam (Cambridge, UK). All other chemicals and reagents were of analytical grade.
6
Immunofluorescence Imaging of AhR
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Cells were fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X-100, and blocked with 2% BSA for 1 h at RT. Samples were incubated with anti-AhR antibody (1:100, Abcam, ab84833) for 1 h at room temperature or overnight at 4°C. Alexa fluor secondary antibody (1:500) was used. DAPI (1 μg/mL) was used to visualize the nucleus. Confocal microscope was used for images.
7
Immunohistochemical Analysis of AhR Expression
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Ileal tissues were fixed in 4% paraformaldehyde overnight, dehydrated, and
embedded in paraffin. Tissue slices (5 µm in thickness) were mounted on
positively charged glass and dewaxed. Ag retrieval was achieved by incubation in
0.01 M sodium citrate buffer, pH 6.0, for 20 min in a boiling steamer. Slides
were then blocked with normal goat serum blocking reagent (Invitrogen) for
30 min and subjected to sequential incubation with anti-AhR (1:50, ab84833,
Abcam) at 4℃ overnight and fluorophore-conjugated secondary Abs at 24℃ for 2 h
and nuclei were visualized with 4[prime],6-diamidino-2-phenylindole (1:2000,
ab104139, Abcam). Between each incubation step, the slides were washed with PBS
three times for 5 min each. Sections were mounted using fluorescent mounting
medium. Confocal images were obtained with a Zeiss (Oberkochen, Germany) LSM 710
imaging system.
embedded in paraffin. Tissue slices (5 µm in thickness) were mounted on
positively charged glass and dewaxed. Ag retrieval was achieved by incubation in
0.01 M sodium citrate buffer, pH 6.0, for 20 min in a boiling steamer. Slides
were then blocked with normal goat serum blocking reagent (Invitrogen) for
30 min and subjected to sequential incubation with anti-AhR (1:50, ab84833,
Abcam) at 4℃ overnight and fluorophore-conjugated secondary Abs at 24℃ for 2 h
and nuclei were visualized with 4[prime],6-diamidino-2-phenylindole (1:2000,
ab104139, Abcam). Between each incubation step, the slides were washed with PBS
three times for 5 min each. Sections were mounted using fluorescent mounting
medium. Confocal images were obtained with a Zeiss (Oberkochen, Germany) LSM 710
imaging system.
8
Histological Analysis of Liver and Intestinal Tissues
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The liver samples were formalin-fixed and sectioned for H&E staining as described.19 (link) Frozen liver sections were stained with Oil red O.19 (link) Formalin-fixed intestinal samples were embedded in paraffin and stained with anti-AHR antibody (ab84833, 1:200; Abcam, Cambridge, MA) and antimyeloperoxidase antibody (ab9535, 1:200; Abcam). All sections were scanned by Nano Zoomer 2.0 RS (Hamamatsu).65 (link)
9
Immunostaining Protocol for AhR in Formalin-Fixed Tumor Sections
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Formalin-fixed tumor sections were stained with hematoxylin and eosin (H&E) or immunostained with an antibody that recognized AhR (cat# ab84833, Abcam, Cambridge, MA, USA) following previously published protocols [28 (link),37 (link)]. All animal studies were carried out according to the procedures approved by the Wayne State University and Virginia Tech Carillon School of Medicine Institutional Animal Care and Use Committees.
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