Ab47943

Manufactured by Abcam

Sourced in United States

Ab47943 is a primary antibody that recognizes a specific target protein. It is intended for use in various laboratory techniques, such as Western blotting, immunohistochemistry, and ELISA, to detect and quantify the target protein in samples. The core function of this product is to bind to the target protein, allowing for its identification and analysis.

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Lab products found in correlation

8 protocols using ab47943

TSLP Expression in Nucleus Pulposus

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Nucleus pulposus tissues were fixed with a 4% paraformaldehyde solution, followed by deparaffinization of paraffin sections. Antigen was repaired under high pressure and at high temperature conditions. The tissues were sealed with normal goat serum. Rabbit polyclonal antibody-TSLP antibody (1:500, ab47943, Abcam Inc., Cambridge, MA) was added and fully mixed with 10 μL of biotin goat anti-rabbit secondary antibody (BA1003, 1:200, BOSTER Biological Technology Co., Ltd., Hubei, China).
Following this, the mixture was used to process the sections over a period of 30 minutes, and then underwent 4 °C overnight inoculation. For developing color diaminobenzidine (DAB) chromogenic (Beijing Cellchip Biotechnology Co., Ltd., Beijing, China) was used. In regards to mouse IgG as blank control, sections were observed under an optical microscope. The cytoplasm was stained using TSLP. Yellow-like particles were regarded as TSLP-positive. From each section, 5 high-power fields (×400) were chosen, and 100 cells were counted in each field. The percentage of TSLP-positive cells/total cells >10% was regarded as positive, while <10% was negative.^{[17 (link)]} Two individuals independently estimated the immunohistochemistry results using the double blind method.

Wang Y., Yi X.D, & Li C.D. (2017). Role of thymic stromal lymphopoietin in the pathogenesis of lumbar disc degeneration. Medicine, 96(30), e7516.

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Immunofluorescence Staining of Lung Tissue

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After dehydration and a blocking treatment, the lung tissue sections were incubated overnight at 4 °C with primary antibodies (TPSAB1 (1:200, 13343-1-AP, Proteintech, Wuhan, China), PAR2 (1:200, ab180953, Abcam, London, UK), GM-CSF (1:200, 17762-1-AP, Proteintech, Wuhan, China), IL-33 (1:200, 66235-1-Ig, Proteintech, Wuhan, China), and TSLP (1:200, ab47943, Abcam, London, UK)). The next day, the sections were treated with a corresponding fluorescently-labeled secondary antibody (Cy3 conjugated goat anti-rabbit IgG (1:300, GB21303, Servicebio, Wuhan, China) or Cy3 conjugated goat anti-mouse IgG (1:300, GB21301, Servicebio)) for 1 h at 25 °C, and were subsequently mounted using a VECTASHIELD mounting medium containing DAPI. Fluorescence images were acquired using the Nikon Eclipse C1 and Nikon DS-U3 systems (Nikon, Tokyo, Japan).

Jia J., Zeng M., Zhu D., Jiao X., Zhang B., Yang R., Feng W, & Zheng X. (2022). An Amide Alkaloid Isolated from Ephedra sinica Ameliorates OVA-Induced Allergic Asthma by Inhibiting Mast Cell Activation and Dendritic Cell Maturation. International Journal of Molecular Sciences, 23(21), 13541.

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Protein Expression Analysis in Cells

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Protein samples of the cells were extracted by lysing the cells with radio immunoprecipitation assay lysis buffer (Beyotime, Shanghai, China). The protein samples were quantified using the BCA Protein Assay Kit (Beyotime) and the same amount of samples were loaded for sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Protein bands on the gel were transferred to a polyvinylidene fluoride membrane and blocked in 5% skim milk for 2 h at room temperature and then the blot was incubated in the specific primary antibodies for TSLP (ab47943), skeletal muscle actin (α-SMA, ab5694), collagen I (ab6308), MAPK7 (ab40809), extracellular signal-regulated kinase 1 (ERK1, ab180163), phospho-ERK1 (p-ERK1, ab24157), p38 (ab7952), p-p38 (ab178867), c-Jun N-terminal kinase 1 (JNK1, ab199380), and p-JNK1 (ab47337), purchased from Abcam (Cambridge, UK), overnight at 4°C. β-actin (ab189073) was used as an endogenous reference. After washing, the blot was incubated in horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. Positive signals were developed using the ECL Plus Western Blotting Substrate (Thermo Scientific). Band densities for each sample were analyzed using ImageJ 1.49 software (National Institutes of Health, Bethesda, MD).

Li L., Tang S, & Tang X. (2016). Thymic Stromal Lymphopoietin Promotes Fibrosis and Activates Mitogen-Activated Protein Kinases in MRC-5 Cells. Medical Science Monitor : International Medical Journal of Experimental and Clinical Research, 22, 2357-2362.

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Multicolor Immunofluorescence Labeling Protocol

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The following primary antibodies were used and combined according to species compatibility: rabbit anti-GluA4C, targeting the C-terminus of GluA4 (1:250, #AB1508, EMD Millipore, Billerica, MA, USA), guinea pig anti-GluA4N, targeting the N-terminus of GluA4 (1:50 for immunohistochemistry #GluA4N-GP-Af640, Frontier Institute Co., Ltd, Hokkaido, Japan), Alexa647-conjugated rat anti-CD34 (1:50 for immunohistochemistry; 1:100 for FACS, #560233, BD Biosciences, San Diego, CA, USA), PE-conjugated rat anti-CD49f-integrin α6 (1:100, #555736, BD Biosciences, San Jose, CA, USA), FITC-conjugated rat anti-Ly-6A/E (Sca-1) (1:100, #11-5981-81, E Bioscience, San Diego, CA, USA), rabbit anti-Loricrin (1:1000, #PRB-145P, BioLegend, Dedham, MA, USA), rabbit anti-TSLP (1:250, #ab47943, Abcam Inc, Cambridge, MA, USA). Alexa Fluor-conjugated secondary antibodies were all obtained from Invitrogen-Molecular Probes, and used at 1:1000 (#A11008 Alexa Fluor 488 Goat Anti-Rabbit (H+L), #A21450 Alexa Fluor 647 goat anti-guinea pig, and #A11005Alexa Fluor 594 Goat anti-mouse (H+L)). In addition to validation by mRNA expression, immunolabeling experimental controls included omission of primary antibodies.

Cabañero D., Irie T., Celorrio M., Trousdale C., Owens D.M., Virley D., Albrecht P.J., Caterina M.J., Rice F.L, & Morón J.A. (2016). Identification of an epidermal keratinocyte AMPA glutamate receptor involved in dermatopathies associated with sensory abnormalities. Pain Reports, 1(3), e573.

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Western Blot Analysis of Protein Expression

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Cells were lysed in lysis buffer (PBS containing 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) supplemented with Complete Protease Inhibitor Cocktail (Roche, Indianapolis, IN) and Phosphatase Inhibitor I and II Cocktails (EMD Millipore, Billerica, MA). Protein concentration was determined by the DC Protein Assay (5000111, Biorad, Hercules, CA) and equal protein was loaded onto 4–20% tris-glycine gels, separated by SDS-PAGE, and transferred to nitrocellulose membranes. Membranes were blocked with 5% bovine serum albumin before being probed with the following antibodies overnight at 4° C: thymic stromal lymphoprotein (TSLP, 1:1000, ab47943, Abcam, Cambridge, United Kingdom), prostaglandin-endoperoxide synthase 2 (PTGS2, 1:1000, Cell Signaling, Danvers, MA), cytochrome P450 1A1 (CYP1A1, 1:1000, sc-393979, Santa Cruz Biotechnology, Dallas, TX), ADAM33 (1:1000, ab137772, Abcam, Cambridge, United Kingdom), PARP (1:1000, Cell Signaling), or α-tubulin (1:5000, Sigma-Aldrich). Membranes were then incubated with appropriate secondary antibody conjugated to horseradish peroxidase (Cell Signaling) for 1 h at room temperature before developing with enhanced chemiluminescent reagent (GE Healthcare, Pittsburgh, PA). For all protein bands, densitometry was analyzed by Image J Software (NIH, Bethesda, MD) and normalized to α-tubulin.

Tripathi P., Deng F., Scruggs A.M., Chen Y, & Huang S.K. (2018). Variation in doses and duration of particulate matter exposure in bronchial epithelial cells results in upregulation of different genes associated with airway disorders. Toxicology in vitro : an international journal published in association with BIBRA, 51, 95-105.

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Immunohistochemical Analysis of OLP, Ulcer, and Hyperkeratosis

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Four-μm formalin-fixed and paraffin-embedded (FFPE) sections of specimens from patients with OLP, ulcer and hyperkeratosis were prepared and stained with a conventional avidin—biotin complex technique as previously described [12 (link)]. The mouse monoclonal antibodies used to analyze the protein expression were anti-CD11c (ab212508; Abcam, Cambridge, MA, USA), and anti-CD68 (ab995; Abcam). Anti-GATA3 (ab199428; Abcam) was a rabbit monoclonal antibody used to analyze the Th2 transcription factor, GATA-3. Rabbit polyclonal antibodies: anti-IFN-γ (500-P32; Peprotech, Rocky Hill, NJ, USA), anti-TSLP (ab47943; Abcam), anti-CRLF2 (ab109626; Abcam), and anti-IL-17 (13082-1-AP; Proteintech, Chicago, IL) were used. Tissue sections were sequentially incubated with primary antibodies for 2.5 h, then with biotinylated anti-mouse IgG or anti-rabbit IgG secondary antibodies (Vector Laboratories, Burlingame, CA, US), avidin—biotin—horseradish peroxidase complex (Vector Laboratories), and 3,3′-diaminobenzidine (Vector Laboratories). Mayer's hematoxylin was used for counterstaining. Photomicrographs were obtained using a light microscope equipped with a digital camera (BZ-9000 series; Keyence, Tokyo, Japan).

Yamauchi M., Moriyama M., Hayashida J.N., Maehara T., Ishiguro N., Kubota K., Furukawa S., Ohta M., Sakamoto M., Tanaka A, & Nakamura S. (2017). Myeloid dendritic cells stimulated by thymic stromal lymphopoietin promote Th2 immune responses and the pathogenesis of oral lichen planus. PLoS ONE, 12(3), e0173017.

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Western Blot Analysis of TSLP and TSLPR

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Tissue samples were homogenized in 400 μL RIPA buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM PMSF, 1 mM EDTA, 1% Triton X-100, 0.5% sodium deoxycholate and 0.1% sodium dodecyl sulfate (SDS)). The protein was separated on SDS-polyacrylamide gels (20 μg per sample) and transferred to a nitrocellulose membrane using standard procedure (Protran, Schleicher & Schuell, city, Germany). The membranes were put in 5% non-fat milk for 1 h at room temperature and then incubated with primary anti-TSLPR antibody (1:1000; sc-83871, Santa Cruz Biotechnology, Dallas, CA, USA) and anti-TSLP antibody (1:10,000; ab47943, Abcam, Cambridge, MA, USA), overnight at 4 °C. After three washes of 5 minutes each in PBS–Tween 20 (0.1%, v/v), membranes were incubated with the secondary antibody (1:5000; AP307P, Millipore, Burlington, CA, USA) conjugated with horseradish peroxidase, anti-rabbit IgG for 2 h at 4 °C and then three 5-min washes were performed. The signal was detected with an enhanced chemiluminescence system according to the manufacturer’s manual (Amersham Pharmacia Biotech, Milan, Italy).

Park C.W., Kim H.J., Choi Y.W., Chung B.Y., Woo S.Y., Song D.K, & Kim H.O. (2017). TRPV3 Channel in Keratinocytes in Scars with Post-Burn Pruritus. International Journal of Molecular Sciences, 18(11), 2425.

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Protein Expression Analysis in Lung Tissue

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A Western blot analysis was performed to detect the expression levels of the TPSAB1 (13343-1-AP, Proteintech, Wuhan, China), PAR2 (ab180953, Abcam, London, UK), GM-CSF (17762-1-AP, Proteintech, Wuhan, China), IL-33 (66235-1-Ig, Proteintech, Wuhan, China), and TSLP (ab47943, Abcam, London, UK). Proteins from the lung tissue were extracted using a mammalian protein extraction kit (Beijing Solarbio, Life Science, Beijing, China) and quantified using a BCA protein assay kit (Solarbio, Life Science, Beijing, China). The protein samples (40 μg) from each group were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene fluoride (PVDF) membrane. After membrane locking with nonfat milk for 1.5 h at room temperature, the membranes were incubated overnight at 4 °C with primary antibodies, followed by a secondary antibody (goat anti-rabbit 925-68071, goat-mouse 925-32210, Li-COR, Lincoln, NE, USA) for 1 h at room temperature. The density of the proteins was quantified using Odyssey (Clx, Li-COR, Lincoln, NE, USA).

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