Vectastain abc kit

Manufactured by Vector Laboratories

Sourced in United States, Canada, United Kingdom, Germany, Japan, France

The Vectastain ABC kit is a product by Vector Laboratories that is used for the detection of specific target antigens in tissue or cell samples. The kit includes reagents necessary for the avidin-biotin complex (ABC) method of immunohistochemistry. The core function of the Vectastain ABC kit is to provide a reliable and sensitive tool for the visualization of target molecules within a sample.

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1 357 protocols using vectastain abc kit

Immunohistochemical Analysis of Tissue Sections

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Immunohistochemical analysis was performed as described earlier^{56 (link)}. Briefly, tissue fragments were fixed in 10% formaldehyde/PBS and embedded in paraffin. Tissues were cut into 5 µm sections and mounted on glass slides and processed with minor modifications as afterprimary antibody incubation and washing, the slides were incubated with biotinylated anti-rabbit IgG (ABC vectastain Kit; Vector Laboratories, Burlingame, CA, USA; 1:200 in PBS) and then treated with avidin-peroxidase (ABC vectastain Kit) and revealed with diaminobenzidine (ABC vectastain Kit), according to the manufacturer’s instructions. Tissues were counterstained with hematoxylin, mounted with DPX (Sigma-Aldrich) and visualized through a Nikon Eclipse-800 microscope. The staining intensity of all these proteins in glandular epithelium, luminal epithelium and stromal compartment were quantified by image analysis software Image-Pro Plus 4.0 (Maryland, USA) and results were expressed as % image analysis score.

Kaushal J.B., Sankhwar P., Kumari S., Popli P., Shukla V., Hussain M.K., Hajela K, & Dwivedi A. (2017). The regulation of Hh/Gli1 signaling cascade involves Gsk3β- mediated mechanism in estrogen-derived endometrial hyperplasia. Scientific Reports, 7, 6557.

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Immunohistochemical Detection of NFIB

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Paraffin sections were obtained from the Department of Pathology at Hannover Medical School. Sections were deparaffinized in xylene, followed by rehydration in an ethanol gradient. Endogenous peroxidase was blocked with 3 % hydrogen peroxide in distilled water. For antigen retrieval, slides were boiled for 5 min in antigen-unmasking solution (Vector Laboratories). After washing with 0.05 % Tween-20 in Tris-buffered saline (TBST) nonspecific binding sides were blocked with blocking solution (1 % BSA with horse serum in TBST; VECTASTAIN ABC kit, Vector Laboratories) for 1 h at room temperature. Afterwards, sections were incubated overnight at 4 °C with primary antibodies against NFIB (1:300, sab1402289, Sigma-Aldrich, St. Louis, MO, USA). After repeated washings with TBST, sections were incubated with biotinylated secondary antibody Ab solution (VECTASTAIN ABC kit, Vector Laboratories) for 30 min and covered with avidin-biotin complex reagent (VECTASTAIN ABC kit, Vector Laboratories) for 1 h at room temperature. Sections were stained in aminoethylcarbazole substrate solution (AEC chromogen kit, Sigma-Aldrich, St. Louis, MO, USA) and counterstained with hematoxylin. Finally, sections were covered with mounting medium (Aquamount, VWR International GmbH, Germany). Ki-67 (1:500, Vector Laboratories, #VP-K451) was stained as previously described [2, 11] .

Roy S., Bantel H., Wandrer F., Schneider A.T., Gautheron J., Vucur M., Tacke F., Trautwein C., Luedde T, & Roderburg C. (2017). miR-1224 inhibits cell proliferation in acute liver failure by targeting the antiapoptotic gene Nfib. Journal of hepatology, 67(5).

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Immunohistochemical Staining of Paraffin Sections

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The paraffin-embedded tissue sections were deparaffinized, treated with 0.01 M citrate buffer (pH 6.0) in a microwave oven for 5 min, chilled at RT for 20 min, and then incubated with 0.3% hydrogen peroxide in DW for 20 min to block endogenous peroxidase. After being washed three times in PBS, the sections were blocked with normal horse serum (VECTASTAIN ABC Kit; Vector Laboratories, Burlingame, CA) and incubated 1 h at RT with N-specific MAb. After rinsing in PBS, the samples were reacted for 45 min at RT with a horse anti-mouse secondary antibody (VECTASTAIN ABC Kit), incubated with avidin-biotin peroxidase complex for 45 min (VECTASTAIN ABC Kit), and developed using the DAB substrate Kit (Vector Laboratories) according to the manufacturer’s instructions. The slides were then counterstained with hematoxylin, dehydrated, cleared with xylene, and mounted on microscope glass slides in mounting buffer and tissue staining was visualized using a microscope.

Lee S., Kim Y, & Lee C. (2015). Isolation and characterization of a Korean porcine epidemic diarrhea virus strain KNU-141112. Virus Research, 208, 215-224.

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Immunohistochemical Analysis of Glutathione S-Transferase-P

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Brains from postnatal day 20 pups were isolated, and sections were generated as aforementioned. For immunohistochemistry, slides were incubated in 5% NGS, 5% BSA, 0.1% Triton X-100 in PBS for 30 min, and then incubated with anti-glutathione-S-transferase-P antibody (1:2,000; MBL Life Science, Japan) in PBS with 5% NGS and 0.2% Triton X-100 overnight. Slides were washed and incubated in biotinylated anti-Rabbit secondary antibody (ABC Vectastain Kit; Vector Laboratories) (1% NGS and 0.2% Triton X-100 in PBS) containing ABC solutions (ABC Vectastain Kit; Vector Laboratories) for 2 h. After washing, slides were stained with 3,3′diaminobenzidine (ImmPACT® DAB Substrate Kit; Vector Laboratories, Burlingame, CA) for 3 min, transferred to PBS, and mounted with DPX (Merck, NJ). Images were obtained and analyzed as aforementioned.

Liu Y., Castano D., Girolamo F., Trigueros-Motos L., Bae H.G., Neo S.P., Oh J., Narayanaswamy P., Torta F., Rye K.A., Jo D.G., Gunaratne J., Jung S., Virgintino D, & Singaraja R.R. (2021). Loss of ABCA8B decreases myelination by reducing oligodendrocyte precursor cells in mice. Journal of Lipid Research, 63(1), 100147.

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Immunohistochemical Detection of F4/80 Macrophages

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Cryostat sections (6 mM thick) were fixed in cold paraformaldehyde 4% in PBS. Immunoperoxidase staining was performed using the avidin-biotin system (ABC Vectastain Kit, Vector Lab., Burlingame, CA, USA) to detect F4/80 antigen. Sections were washed in PBS and incubated with 5% skim milk, 0.01% Triton X-100, and then treated with avidin–biotin blocking solution (Vector Laboratories). After overnight incubation with a rat anti-mouse F4/80 antibody (1:5) (Tonbo Biosciences, San Diego, CA, USA) at 4 °C in a humidified chamber, sections were incubated with biotinylated anti-rat IgG (1:250, Vector Lab.). Endogenous peroxidase activity was blocked by treatment with 0.3% H₂O₂ in methanol for 30 min. Then, the reaction was amplified using ABC Vectastain Kit and the 3-3′diaminobenzidine-H₂O₂ (DAB Substrate Kit, Vector Lab.) was used as peroxidase substrate. Sections were counterstained with hematoxylin.

Carvajal G., Brukman N.G., Weigel Muñoz M., Battistone M.A., Guazzone V.A., Ikawa M., Haruhiko M., Lustig L., Breton S, & Cuasnicu P.S. (2018). Impaired male fertility and abnormal epididymal epithelium differentiation in mice lacking CRISP1 and CRISP4. Scientific Reports, 8, 17531.

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Histological Analysis of Liver Tissues

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Liver tissues were fixed in Bouin's solution (Sigma, St. Louis, USA), embedded in paraffin, sectioned at 6 μm, and stained using hematoxylin and eosin (H&E; Sigma, St. Louis, USA) and Masson's trichrome stain (Trichome stain kit; ScyTek Laboratories, West Logan, USA) using standard protocols [20 (link), 21 (link)]. Nuclear counterstaining was performed using hematoxylin, and the samples were examined using light microscopy (Nikon, Tokyo, Japan). For Slc25a15 immunostaining, the samples were incubated first with 1 : 300 dilutions of the anti-Slc25a15 antibody (Abcam, Cambridge, UK) and then with a biotinylated anti-mouse IgG (Vectastain ABC Kit; Vector Labs, Burlingame, USA). The sections were incubated with the avidin–biotin–peroxidase complex (Vectastain ABC Kit; Vector Labs, Burlingame, USA) and DAB. The Celleste image analysis software (Thermo Fisher Scientific, Waltham, USA) was used to count the number of immunoreactive cells.

Lee Y.M., Choi D.H., Cheon M.W., Kim J.G., Kim J.S., Shin M.G., Kim H.R, & Youn D. (2022). Changes in Mitochondria-Related Gene Expression upon Acupuncture at LR3 in the D-Galactosamine-Induced Liver Damage Rat Model. Evidence-based Complementary and Alternative Medicine : eCAM, 2022, 3294273.

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Immunohistochemical Analysis of SHH Expression

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For IHC, deparaffinized sections were pretreated with 10 mM sodium citrate buffer for antigen unmasking (pH 6.0, boiling temperature, 30 min), blocked in normal serum (Vectastain ABC kit, Vector Laboratories, Inc. Burlingame, CA), incubated with primary antibodies at 4oC overnight, rinsed, and incubated with secondary antibody (Vectastain ABC kit). Signals were amplified using Vectastain ABC kit per manufacturer’s instruction. Targeted protein was visualized using diaminobenzidine as substrate. The results were interpreted by two independent pathologists who were blinded to the specific diagnosis and prognosis for each case, and were scored by a semi-quantitative method in which staining of more than 10 % of the tumor cells were considered positive. The staining intensity was scored as “negative”,“weak staining”, “moderated staining” and “strong staining”. Low SHH expression was determined by negative and weak staining, and high SHH expression was determined by moderate and strong staining.

Ertao Z., Jianhui C., Chuangqi C., Changjiang Q., Sile C., Yulong H., Hui W, & Shirong C. (2016). Autocrine Sonic hedgehog signaling promotes gastric cancer proliferation through induction of phospholipase Cγ1 and the ERK1/2 pathway. Journal of Experimental & Clinical Cancer Research : CR, 35, 63.

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Immunohistochemical Analysis of Placental IL-6 and IGFBP-1

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For term placenta, paraffin-embedded sections were subjected to the avidin-biotin-peroxidase complex method using a Vectastain ABC kit (Vector Laboratories, Inc., Burlingame, CA) as described previously with some modifications [30 (link)]. Briefly, formalin-fixed, paraffin-embedded sections (5μm) were deparaffinized, hydrated, and blocked with 5% normal donkey serum. The slides were then incubated overnight at 4°C with a polyclonal antibody to IL-6 (Abcam Inc., Cambridge, MA) or a mouse monoclonal antibody to IGFBP-1 at 1:100. The slides were then incubated with corresponding secondary biotinylated goat IgG according to the manufacturer’s instructions (Vectastain ABC kit; Vector Laboratories, Burlingame, CA). Peroxidase activity was developed with Nova Red or DAB solution (Vector Laboratories, Inc., Burlingame, CA). For preterm placenta, frozen sections were fixed in pre-cooled methanol at -20°C for 10 min. Sections were processed for immunohistochemistry as described for term placenta. Tissue sections from at least 3 different placentas were used for each group.

Devi Y.S., DeVine M., DeKuiper J., Ferguson S, & Fazleabas A.T. (2015). Inhibition of IL-6 Signaling Pathway by Curcumin in Uterine Decidual Cells. PLoS ONE, 10(5), e0125627.

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Quantification of BDNF and ΔFosB Immunoreactivity

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Sections were washed in 0.05 M potassium phosphate-buffered saline (KPBS), then blocked for 1 h in 10% normal donkey serum and 0.4% Triton X-100 in 0.05 M KPBS, and incubated with primary antibody: BDNF (AB1779SP, 1:3,000 dilution; Millipore; Temecula, CA) or FosB (SC-48, 1:5,000 dilution; Santa Cruz Biotechnology, Inc.; Santa Cruz, CA). The FosB antibody used here targets the N terminal of the FosB protein contained in both FosB and ΔFosB isoforms. However in this experiment, FosB-like labeling would primarily capture accumulation of ΔFosB; FosB expression is transient and only ΔFosB persists for days after stimulation (Perrotti et al., 2004 (link)). Following incubation with primary antibody for 48 h at 4°C, slides were washed in 0.05 M KPBS and incubated for 1 h in biotin-conjugated goat anti-rabbit serum (1:200 dilution in blocking solution, Vectastain ABC kit; Vector Laboratories; Burlingame, CA). After washing in 0.05 M KPBS, sections were incubated with avidin–biotin–peroxidase complex (Vectastain ABC kit) for 1 h, then washed again and developed using DAB chromogen with nickel intensification (DAB Peroxidase Substrate kit, Vector Laboratories). After dehydration in graded concentrations of ethanol and xylene, coverslips were applied.

Wang J., Bastle R.M., Bass C.E., Hammer RP J.r., Neisewander J.L, & Nikulina E.M. (2016). Overexpression of BDNF in the ventral tegmental area enhances binge cocaine self-administration in rats exposed to repeated social defeat. Neuropharmacology, 109, 121-130.

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Quantification of Cellular Proliferation

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Tumors were fixed in 10% neutral buffered formalin for 24 h followed by 70% ethanol and were then embedded in paraffin. Sections (∼5 µm) were cut and stained with hematoxylin and eosin (H&E). IHC for Ki-67 expression was carried out to determine the number of proliferating cells. Briefly, sections were deparaffinized, hydrated, and antigen retrieved by using 10 mM citrate buffer (pH 6.0). Sections were first blocked with 3% H₂O₂ and then with 1.5% blocking serum (Vectastain ABC kit, Vector laboratories; Burlingame, CA). Sections were then incubated with the anti-Ki-67 antibody for 30 min at room temperature. Following two washes in PBS, sections were incubated with biotinylated secondary antibody and then with the enzyme reagent (Vectastain ABC kit; Vector laboratories). Sections were stained with diaminobenzidine (DAB) and counterstained using Hematoxylin nuclear stain (Vector laboratories; #H-3401). Permanent mounting media was added and Ki-67 staining was visualized and captured by using an Eclipse E-400 microscope (Nikon Instruments, Melville, NY). In each slide, 5 different fields were visualized for Ki-67 stained cells and quantified by using the Image-J software.

Mathur A., Abd Elmageed Z.Y., Liu X., Kostochka M.L., Zhang H., Abdel-Mageed A.B, & Mondal D. (2014). Subverting ER-Stress towards Apoptosis by Nelfinavir and Curcumin Coexposure Augments Docetaxel Efficacy in Castration Resistant Prostate Cancer Cells. PLoS ONE, 9(8), e103109.

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