Abc elite kit

Manufactured by Vector Laboratories

Sourced in United States, Canada, United Kingdom, Japan

The ABC Elite kit is a comprehensive laboratory tool designed for versatile applications. It includes a range of high-quality components and accessories to support various experimental procedures. The core function of the kit is to provide researchers with a reliable and efficient solution for their laboratory needs.

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319 protocols using abc elite kit

Quantifying FRA and Kisspeptin Neurons in ARC

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Sections were incubated with the anti-FRA rabbit antibody (K-25; Santa Cruz Biotechnology, Santa Cruz, CA, USA), at 1:2,000 for 40 h, biotinylated anti-rabbit goat IgG (Vector Laboratories, Burlingame, CA, USA) at 1:600 for 90 min, and avidin-biotin complex solution at 1:100 for 1 h (Elite ABC kit, Vector Laboratories). A solution of nickel sulphate (25 mg/ml), 3,3’-diaminobenzidine-HCl (DAB, 0.2 mg/ml), and 0.03% H₂O₂ (Ni-DAB) was used as the chromogen. Then, sections were then incubated with anti-mouse Kp-10 antibody raised in rabbits (A.C. 564), at 1:10,000 for 40 h, biotinylated anti-rabbit goat IgG (Vector Laboratories) at 1:600 for 90 min, and Elite ABC kit at 1:100 for 1 h. DAB solution was used as chromogen. Brain sections were blindly analyzed for experimental groups under a light microscope with an image analysis system (Motic). The number of FRA-, kisspeptin-, and FRA/kisspeptin-immunoreactive (ir) neurons was quantified bilaterally in the ARC (−1.88 to −4.2 from Bregma), according to Paxinos and Watson (24 ).

Nicola A.C., Ferreira L.B., Mata M.M., Vilhena-Franco T., Leite C.M., Martins A.B., Antunes-Rodrigues J., Poletini M.O, & Dornelles R.C. (2021). Vasopressinergic Activity of the Suprachiasmatic Nucleus and mRNA Expression of Clock Genes in the Hypothalamus-Pituitary-Gonadal Axis in Female Aging. Frontiers in Endocrinology, 12, 652733.

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Quantification of DNA Damage in Tumor Sections

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All in vitro studies were performed from a minimum of three experimental replicates. Staining was performed on formalin-fixed paraffin embedded tumor sections (3-5 µm) using the Vectastain elite avidin-biotin complex kit (Vector Laboratories, Burlingame, CA, USA). Briefly, after antigen retrieval with 10 mM sodium citrate (pH 6.5) paraffin sections were incubated with rabbit anti-γ-H2AX monoclonal antibody (Ser139) (Novus Biologicals, Littleton, CO) in blocking solution (1% serum in PBS plus 0.4% Triton X-100, ABC Elite Kit, Vector Laboratories) at 4ºC overnight. All sections were processed with the ABC Elite Kit (Vector Laboratories) per manufacturer’s recommendation. The immunoreactivity was visualized using peroxidase-DAB (3,3′-diaminobenzidine). All sections were counterstained with Mayer’s hematoxylin, dehydrated and cover slipped. Negative controls with no primary antibody were used to assess nonspecific staining. Two tumors per time point, a minimum of three sections per tumor and 20 fields per section were quantified.

Bakewell S.J., Carie A., Costich T.L., Sethuraman J., Semple J.E., Sullivan B., Martinez G.V., Dominguez-Viqueira W, & Sill K.N. (2017). Imaging the Delivery of Drug-loaded, Iron-stabilized Micelles. Nanomedicine : nanotechnology, biology, and medicine, 13(4), 1353-1362.

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Histological Analysis of Atherosclerotic Lesions

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Hearts were collected and fixated in phosphate-buffered 4% formaldehyde, embedded in paraffin after dehydration in 70-100% ethanol and cross-sectioned (5 μm) perpendicular to the axis of the aorta throughout the aortic root area, starting from the appearance of open aortic valve leaflets. Per mouse, 4 sections with 50 μm intervals were used for atherosclerosis measurements. Sections were stained with haematoxylin-phloxine-saffron for histological analysis. Lesions were categorized by severity according to the guidelines of the American Heart Association adapted for mice [21] (link). Sirius Red staining was used to quantify the collagen area. Monoclonal mouse antibody M0851 (1:800; Dako, Heverlee, The Netherlands) against smooth muscle cell actin was used to quantify the smooth muscle cell area. Rat monoclonal anti-mouse MAC-3 antibody (1:1000; BD Pharmingen, San Diego, CA, USA) was used to quantify the macrophage area. Immunostainings were amplified using Vector Laboratories Elite ABC kit (Vector Laboratories Inc., Burlingame, CA, USA) and the immune-peroxidase complex was visualized with Nova Red (Vector Laboratories Inc., Burlingame, CA, USA). Lesion area and composition were analyzed using Image J software. The stability index was calculated by dividing the relative collagen and smooth muscle cell area by the relative area of macrophages within the same lesion.

Liu C., Li Z., Song Z., Fan X., Shao H., Schönke M., Boon M.R., Rensen P.C.N, & Wang Y. (2022). Choline and butyrate beneficially modulate the gut microbiome without affecting atherosclerosis in APOE*3-Leiden.CETP mice. Atherosclerosis, 362.

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Adipose Tissue Histological Analysis

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Formalin‐fixed interscapular BAT (iBAT), subcutaneous WAT (sWAT), and gonadal WAT (gWAT) were dehydrated in 70% EtOH, embedded in paraffin, and cut into 5‐μm sections. Sections were stained with hematoxylin and eosin (H&E) using standard protocols. UCP1 staining was performed as previously described (Berbee et al, 2015). In short, sections were treated with 3% H₂O₂ for 30 min and boiled in citrate buffer (10 mM, pH 6) for 10 min. Slides were blocked with 1.3% normal goat serum, incubated overnight at 4°C with rabbit monoclonal anti‐UCP1 antibody (1:400, Abcam) followed by 1‐h incubation with biotinylated goat α‐rabbit secondary antibody (Vector Labs). Immunostaining was amplified using Vector Laboratories Elite ABC kit (Vector Labs) and visualized with Nova Red (Vector Labs). Counterstaining was performed with hematoxylin. All sections were digitalized with Philips Digital Pathology Solutions (PHILIPS Electronics) for morphological measurement. White adipocyte size, iBAT lipid droplet content, and UCP1 expression (relative UCP1 staining per area) were quantified using ImageJ software (Version 1.50).

Schilperoort M., van Dam A.D., Hoeke G., Shabalina I.G., Okolo A., Hanyaloglu A.C., Dib L.H., Mol I.M., Caengprasath N., Chan Y., Damak S., Miller A.R., Coskun T., Shimpukade B., Ulven T., Kooijman S., Rensen P.C, & Christian M. (2018). The GPR120 agonist TUG‐891 promotes metabolic health by stimulating mitochondrial respiration in brown fat. EMBO Molecular Medicine, 10(3), e8047.

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Immunohistochemical Analysis of PD-L1 Expression

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Formalin-fixed and paraffin-embedded specimens were sectioned at 5 μm thickness and mounted on glass slides. The PD-L1 (clone 5H1) antibody was obtained from Lieping Chen (12 (link)). In brief, antigen retrieval was achieved with citrate buffer. Anti-PD-L1/B7-H1 murine IgG (1:1000) was applied on tissue sections overnight at 4°C. After washing with TBS (0.05 M Tris base, 0.9% NaCl, pH 8.4), tissue sections were incubated with biotinylated anti-mouse IgG (1:100; BA-2001, Vector Laboratories) followed by incubation with Elite ABC kit for 30 min at room temperature. Antibody binding was detected with the TSA Biotin System (NEL700A001KT, PerkinElmer) and DAB (DAKO, K3468). Murine PD-L1 staining was performed using the polyclonal antibody against mouse B7-H1/PD-L1 antibody (1:100, R&D Systems). Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and KIT immunostaining (1:200, D13A2; Cell Signaling Technology) were performed as described (24 (link)). Slides were analyzed and imaged on the Axio Imager 2 wide-field microscope (Zeiss).

Seifert A.M., Zeng S., Zhang J.Q., Kim T.S., Cohen N.A., Beckman M.J., Medina B.D., Maltbaek J.H., Loo J.K., Crawley M.H., Rossi F., Besmer P., Antonescu C.R, & DeMatteo R.P. (2016). PD-1/PD-L1 blockade enhances T cell activity and antitumor efficacy of imatinib in gastrointestinal stromal tumors. Clinical cancer research : an official journal of the American Association for Cancer Research, 23(2), 454-465.

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Cytological and Histopathological Analysis of Cell Samples

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Cytospin preparations from peripheral blood or bone marrow cells stained with May-Grunwald-Giemsa as described previously (65 (link)). Tissue samples were fixed formalin, dehydrated and embedded in paraffin. Sectioned slides were rehydrated and followed by standard H&E staining protocol. For IHC, antigen was retrieved by boiling slides in 10mM sodium citrate buffer at 90–100°C for 20 minutes and then Cooled down to room temperature. After washing twice with PBST, slides were incubated in methanol with 3% H₂O₂ for 20 minutes followed by blocking with 5% goat serum. Primary antibody used was c-Kit (CD117) (Biolegend, Cat# 105802). Detection was performed with the Elite ABC Kit and DAB Substrate (Vector Laboratories), followed by hematoxylin counterstaining (Sigma). Reticulin and trichrome staining of BM, spleen or liver sections were performed by the Molecular Pathology Core facility at UTSW.

Gu Z., Liu Y., Cai F., Patrick M., Zmajkovic J., Cao H., Zhang Y., Tasdogan A., Chen M., Qi L., Liu X., Li K., Lyu J., Dickerson K.E., Chen W., Ni M., Merritt M.E., Morrison S.J., Skoda R.C., DeBerardinis R.J, & Xu J. (2019). Loss of EZH2 Reprograms BCAA Metabolism to Drive Leukemic Transformation. Cancer discovery, 9(9), 1228-1247.

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FosB/ΔFosB Immunohistochemistry in Mice

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One hour after the last 6-Hz test, mice were deeply anesthetized with isoflurane for about 10 s and perfused transcardially with phosphate buffered saline (PBS, pH 7.4), followed by Zamboni’s fixative (pH 6.9). Brains were removed and postfixed at 4 °C in the same fixative for 24 h [18 (link),19 (link)]. Tissues were transferred to 15% and 30% sucrose solutions, and then horizontal sections (50 μm thickness) were cut using a freezing-sliding microtome (Leica SM2000 R; Leica, Nussloch, Germany).
Sections were washed three times in PBS, treated with 3% H₂O₂ in PBS (10 min), and blocked for 1 h in PBS containing 5% normal goat serum and 0.1% Triton X-100. The sections were subsequently incubated overnight at 4 °C with rabbit polyclonal anti-FosB/ΔFosB (H-75, sc-7203, Santa Cruz Biotechnology, CA, USA; 1:250). The next day, the sections were incubated for 1 h with a biotinylated anti-rabbit secondary antibody (Vector Laboratories, Burlingame, CA, USA; 1:200). After washing, the brain sections were incubated for 1 h with the avidin-biotin-peroxidase complex (Elite ABC Kit; Vector Laboratories). The immunostaining was developed in 0.05% 3,3-diaminobenzidine tetrahydrochloride for 5 min (Sigma-Aldrich, Milan, Italy) by adding 0.03% H₂O₂.

Costa A.M., Senn L., Anceschi L., Brighenti V., Pellati F, & Biagini G. (2021). Antiseizure Effects of Fully Characterized Non-Psychoactive Cannabis sativa L. Extracts in the Repeated 6-Hz Corneal Stimulation Test. Pharmaceuticals, 14(12), 1259.

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Quantifying Tumor Cell Proliferation

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The proliferation of the PANC-1 tumor cells was determined by the expression of proliferating cell nuclear antigen (PCNA) using immunohistochemical staining. In brief, tumors were excised from each mouse and weighed at the end of the experiment. Tumor tissues were fixed in buffered formalin for 24 h and then with ethanol for 48 h. Paraffin blocks of tumor tissues were prepared and paraffin sections of tumor tissues were processed for immunohistochemical staining. The sections were incubated with PCNA antibody (MAB424; Millipore Corp.) for 1 h at room temperature. The sections were then incubated with a biotinylated secondary antibody for 30 min followed by incubation with horseradish peroxidase conjugated-avidin solution for 30 min using the Elite ABC kit (PK-6100; Vector Laboratories, Burlingame, CA, USA). PCNA staining in the tumor cells (brown color in nucleus) was examined under a microscope (Nikon Optiphot; Nikon). At least 1,000 cells were counted for each section.

HUANG H., CAO K., MALIK S., ZHANG Q., LI D., CHANG R., WANG H., LIN W., VAN DOREN J., ZHANG K., DU Z, & ZHENG X. (2015). Combination of 12-O-tetradecanoylphorbol-13-acetate with diethyldithiocarbamate markedly inhibits pancreatic cancer cell growth in 3D culture and in immunodeficient mice. International Journal of Molecular Medicine, 35(6), 1617-1624.

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Immunodetection of Runx2 and Osterix in Bone Tissue

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Runx2 and osterix immunohistochemistry was performed according to the peroxidase-antiperoxidase (PAP) method as we reported in our previous study (Evan et al, 2008 (link)). The paraffin slides were deparaffinized and rehydrated through graded alcohols and blocked for nonspecific protein binding using 20% normal goat serum in 0.1 mol/L phosphate-buffered saline (PBS) at room temperature for 2 hours. A recombinant human Runx2/CBFA1 primary antibody derived from a rat monoclonal IgG2B clone was purchased from R and D Systems (Minneapolis, MN) and used at a 1:25 dilution. A human Osterix primary antibody was purchased from Santa Cruz Biotechnology, Inc (Dallas, TX) and used at a 1:35 dilution. The secondary antibody was biotinylated goat anti-mouse IgG (Sigma, St. Louis, MO). Reactivity was detected with the ELITE ABC kit (Vector Labs, Burlingame, CA) and DAB (3,3-diaminobenzidine tetrahydrochloride). DAB in Tris-HCl buffer was used as the chromogen. All sections were covered with DAB solution (0.05 mol/L Tris-HCl, 0.001% hydrogen peroxide, and 0.10% DAB), and incubated for approximately 10 minutes. Finally, each slide was rinsed well with buffer and lightly counterstained with hematoxylin. Controls were performed with the elimination of the primary antibody and showed no staining.

Evan A.P., Worcester E.M., Williams JC J.r., Sommer A.J., Lingeman J.E., Phillips C.L, & Coe F.L. (2015). BIOPSY PROVEN MEDULLARY SPONGE KIDNEY: Clinical findings, histopathology, and role of osteogenesis in stone and plaque formation. Anatomical record (Hoboken, N.J. : 2007), 298(5), 865-877.

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Immunohistochemical Analysis of Breast Cancer

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Immunohistochemical analysis of human breast cancer tissues in TMA was performed according to the avidin-biotinylated-HRP complex (ABC) method using an Elite ABC kit (Vector Laboratories, Burlingame, CA, USA). The TMA slides were transferred to a xylene chamber and dipped in a graded ethanol series. After antigen retrieval in 10 mM sodium citrate buffer (pH 6.0) for 15 min, the slides were transferred to 3% hydrogen peroxide in methanol to quench the endogenous peroxidase activity. To block non-specific binding sites, slides were incubated in diluted normal goat serum at 24°C for 30 min. The slides were then incubated overnight at 4°C with anti-GLI1 (ab217326, 1:1000). The slides were washed twice with TBS and incubated with biotinylated goat anti-rabbit IgG (1:200) for 30 min at 24°C. After rinsing the slides in TBS, they were incubated with ABC reagent for 30 min. Color development was achieved by applying DAB solution for 2 min. After washing in distilled water, the slides were counterstained with hematoxylin, dehydrated with ethanol and xylene, and coverslipped using a mounting solution. Images were captured using a Zeiss Axiovert 200 microscope (Zeiss, Germany) equipped with a PAXCAM microscope camera.

Lee S.H., Lee J.S., Park J.H., Yoon S., Lee K.Y, & Kim H.S. (2022). Glycolytic Metabolic Remodeling by the Truncate of Glioma-Associated Oncogene Homolog 1 in Triple-Negative Breast Cancer Cells. Journal of Cancer, 13(10), 3031-3043.

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