Immunohistochemistry was performed on 4-μm-thick sections of paraffin-embedded specimens using anti-Ki67 (ab15580, abcam) at 4 degrees in a moist chamber overnight. Then treated with the biotin-labelled secondary antibody, a VECTASTAIN Elite ABC peroxidase kit (PL-6100, Vector laboratories), followed by colour development with DAB. 40× images were acquired with Ni-E brightfield microscope (Nikon, Tokyo, Japan)
Vectastain elite abc peroxidase kit
Manufactured by Vector Laboratories
Sourced in United States, United Kingdom
The Vectastain Elite ABC peroxidase kit is a product manufactured by Vector Laboratories. It is a reagent system designed for the detection of antigens in tissue sections or cell preparations using an avidin-biotin-peroxidase complex (ABC) method.
Automatically generated - may contain errors
Lab products found in correlation
Fluorescence microscope, by Nikon (5 mentions)
Ab108422, by Abcam (4 mentions)
Ab735, by Abcam (4 mentions)
Hoechst 33342, by Thermo Fisher Scientific (4 mentions)
Hoxb13, by Santa Cruz Biotechnology (3 mentions)
Sc 1488, by Santa Cruz Biotechnology (3 mentions)
Immpact dab kit, by Vector Laboratories (3 mentions)
Dab substrate, by Merck Group (2 mentions)
Chromogranin a, by Santa Cruz Biotechnology (2 mentions)
Mayer s hematoxylin, by Merck Group (2 mentions)
Enhanced hrp dab chromogenic kit, by Tiangen Biotech (2 mentions)
Ckx41 microscope, by Olympus (2 mentions)
Ox 38, by BD (2 mentions)
Aec substrate 3 amino 9 ethylcarbazole, by Thermo Fisher Scientific (2 mentions)
Chromogranin a chga sc 28333, by Santa Cruz Biotechnology (2 mentions)
Tsa biotin tyramide kit, by PerkinElmer (2 mentions)
Biotinylated goat anti human igg, by Jackson ImmunoResearch (2 mentions)
Streptavidin alexafluor 568, by Thermo Fisher Scientific (2 mentions)
A15278, by ABclonal (2 mentions)
Synaptophysin syp, by BD (2 mentions)
63 protocols using vectastain elite abc peroxidase kit
1
Immunohistochemistry for Ki67 in Paraffin Sections
2
Immunohistochemical Staining Protocol
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Free floating sections (thickness; 40μm) were stained using standard immuno-histochemical procedures as described earlier [21, 22 (link)]. Briefly, after quenching with 3% hydrogen peroxide (H2O2) and 10% methanol for 5 min sections were pre-incubated in 2% BSA and normal horse serum (NGS; Vector Laboratories, Burlingame, CA) and 0.3% Triton X-100 for 60 min followed by incubation with primary antibodies overnight under cold conditions, followed by incubation for 2 h with the biotinylated secondary antibody (1 : 200, Vector Laboratories) the next day. Vectastain Elite ABC peroxidase kit (Vector Laboratories) was used for visualization using 0.06% diaminobenzidine and H2O2.
3
Visualization of Phosphorylated EGFR in Embryos
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For visualization of skin cells with phosphorylated EGFR, embryos were fixed overnight at 4°C in ethanol–acetic acid–formaldehyde fixative (40% ethanol, 5% acetic acid, and 10% formalin), and washed extensively in 0.1% PBS–Tween-20. Quenching of endogenous peroxidases was achieved by incubating the embryos in 3% H2O2 for 5 min. Blocking was performed in blocking buffer (2% BSA, 1% DMSO, in 0.1% PBS–Tween-20) for at least 2 h, and embryos were incubated in primary antibody (polyclonal anti-pEGFR [Tyr1068], RRID AB_2533754, Thermo Fisher Scientific, cat. no. 44-788G) at a 1:50 dilution overnight at 4°C, and then with a biotinylated goat anti-rabbit secondary at 1:250 (RRID AB_2313606, Vector Laboratories, cat. no. BA-1000) following extensive washing. ABC amplification was performed following the manufacturer’s instructions (Vectastain Elite ABC Peroxidase kit, Vector Labs), and embryos were incubated in DAB substrate (Sigma-Aldrich) and H2O2 until development of a signal. Embryos were then post-fixed in 4% PFA, transferred to methanol, and cleared in benzyl alcohol/benzyl benzoate (1:2; both Sigma-Aldrich) before imaging using a Zeiss Axioplan 2 microscope.
4
Immunohistochemical Analysis of Immune Cells
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Tissue sections were deparaffinized in xylene and rehydrated with decreasing concentrations of alcohol. Subsequently, endogenous peroxide was blocked with hydrogen peroxide, and antigen retrieval was achieved by treating sections with 0.01 m sodium citrate, pH 6.0 for 1 min in a pressure cooker. After blocking with universal blocking solution (ZYMED Laboratories, San Francisco, CA, USA), tissue sections were stained with the designated primary antibodies. A Vectastain Elite ABC Peroxidase kit or Universal ImmPRESS kit (Vector Laboratories, Burlingame, CA, USA) were used for secondary antibodies, and visualization was performed using 3-amino-9-ethylcarbazole as a substrate (ZYMED Laboratories, San Francisco, CA, USA). Sections were then stained with hematoxylin for counterstaining and mounted using VectaMount AQ Aqueous Mounting Medium (Cat-No. H-5501; Vector Laboratories). Myeloperoxidase-positive (Abcam), F4/80-positive (Santa Cruz, 377009), and LY6C-positive cells (Abcam, ab15627) were counted in stained sections in six randomly chosen fields (×200), and bars are indicated standard errors of the means.
5
Immunohistochemistry Analysis of HIV-PML Brain
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Formalin-fixed, paraffin-embedded sections of HIV-PML brain tissues were kindly provided by Dr. Susan Morgello, Manhattan HIV Brain Bank, NNTC, sectioned at 4μM in thickness. Immunohistochemistry was performed using the avidin–biotin–peroxidase complex system (Vectastain Elite ABC Peroxidase Kit, Vector Laboratories, Inc., Burlingame, CA). After deparaffinization with xylene, rehydration, non-enzymatic antigen retrieval was performed in 0.01M citrate (pH 6.0) for 30 min at 95°C followed by cooling for 20 min. All sections were then quenched for endogenous peroxidase in methanol/3% H2O2 for 20 min. After blocking with serum/BSA, primary antibodies were added and incubated overnight in a humidified chamber. Sections were then incubated with the appropriate biotinylated secondary antibody followed by detection with DAB (0.02% diaminobenzidine and 0.005% hydrogen peroxide), counterstaining with hematoxylin, and mounting with Permount.
6
Immunohistochemical Staining Protocol
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The tissue samples were fixed in 10% formalin, embedded in paraffin, cut into 4-µm sections, and stained with hematoxylin and eosin following a routine protocol. The sections were deparaffinized with xylosine, followed by rehydration in graded ethanol. Endogenous peroxidase activity was blocked with 3% by volume hydrogen peroxidase in methanol. Antigen retrieval was performed by microwave irradiation in 10 mmol/L sodium citrate (pH 6.0), followed by washing in phosphate-buffered saline (PBS) and staining using a Vectastain Elite ABC peroxidase kit (Vector Laboratories, Peterborough, UK). In addition, to avoid non-specific binding, an avidin/biotin blocking kit was used. The block was then removed and the slides were incubated overnight with a primary antibody. Binding was visualized by incubation with the peroxidase substrate 3-amino-9-ethylcarbazole and counterstained with 10% hematoxylin. Negative controls were obtained by blocking the primary antibody with the corresponding specific peptide. Immunostained sections were examined using an Olympus BH2 microscope (Tokyo, Japan) [23 (link)].
7
Immunohistochemical Analysis of Prostate Cancer
8
Immunohistochemical Analysis of Xenograft Tumors
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Immunohistochemistry was performed according to standard methods. Paraffin-embedded xenograft tumor tissues were cut into 3-μm sections, deparaffinized, and rehydrated in different percentages (100, 95, 85, 75, and 65%) of ethanol. For antigen retrieval and to quench endogenous peroxidases, sections were incubated with 10 mM citric buffer (pH 6.0) and BLOXALL Blocking Solution (Vector Laboratories; Burlingame, CA, USA), respectively. The slides were incubated overnight at 4°C with primary antibodies against p-p65, cleaved Caspase3, and Ki-67. Detection was performed using the VECTASTAIN Elite ABC-Peroxidase Kit (Vector Laboratories) and an Enhanced HRP–DAB Chromogenic Kit (TIANGEN Biotech Co., Ltd; Beijing, China) according to the manufacturer’s instructions, followed by counterstaining with Mayer’s hematoxylin (Sigma–Aldrich). The slides were dehydrated with different concentrations of ethanol (65, 75, 85, 95, and 100%) and then mounted in Permount Mounting Medium (Fisher Scientific). Images were captured at ×400 magnification under a CKX41 microscope (Olympus, Tokyo, Japan).
9
Immunohistochemical Analysis of Prostate Cancer
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Immunostaining was performed using Vectastain elite ABC peroxidase kit (Vector Laboratories, Burlingame, CA) as described previously.11 (link) Primary antibodies of YAP1 and Chromogranin A (CHGA) were purchased from Santa Cruz Biotechnology (sc-101199 and sc-1488 respectively, Dallas, TX), Synaptophysin (SYP) (611880, BD biosciences, San Jose, CA), p63 and FOXA2 (ab735 and ab108422, respectively, Abcam, Cambridge, MA). The tissue sections were counterstained, mounted, and imaged with a Zeiss microscope (White Plains, NY). The percentage of cells stained was evaluated on a scale of 1+ (1–25%), 2+ (25–50%), 3+ (50–75%), and 4+ (75–100%) and the intensity of expression, 0 (negative), 1+ (weak), 2+ (moderate), and 3+ (strong). To assess the correlation between the expression of YAP1 and the Gleason score of tumor samples, immunohistochemistry results were further evaluated by a semiquantitative H-score in a blinded manner. For immunofluorescence staining, YAP1 was co-stained with cytokeratin 5 (CK5) or 14 (CK14) (904801 and 905501 respectively, BioLegend, San Diego, CA) and imaged with a Nikon fluorescence microscope (Melville, NY).
10
Immunostaining of Epithelial Cells
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For immunostaining, epithelial cells were cultured in 4-well chamber slides at 37℃ in DMEM/F12 culture medium (Invitrogen) supplemented with 1% penicillin and streptomycin (Sigma-Aldrich), 10% FCS (Invitrogen) and L-glutamine (Invitrogen) in a 5% CO2 atmosphere. At confluency, the slides were washed with PBS, fixed with 5% formalin, and stored at 4℃. Staining for cytokeratin was performed using a Vectastain Elite ABC peroxidase kit (Vector Laboratories). To avoid non-specific binding, an avidin/biotin blocking kit (Vector Laboratories) was used. After removing the block, the slides were incubated for 2 hours at room temperature in primary antibody (monoclonal anti-pan cytokeratin antibody clone C-11, Sigma-Aldrich) at an appropriate dilution using antibody diluent medium (Dakocytomation Ltd., Cambridgeshire, UK) and 250 mL of biotin per milliliter of diluted antibody. Binding was visualized by incubation with DAB substrate (Vector Laboratories), and the slides were then counterstained with hematoxylin and mounted with DPX (VWR International, Lutterworth, UK).
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