Human breast tumor tissue array (BC081116d) with known ERα status was obtained from US Biomax (Derwood, MD). The sections were deparaffinized and rehydrated through graded ethanol. Antigen retrieval was performed using citrate buffer pH 6.0 prior to incubation with 3% H2O2 to reduce endogenous peroxidase activity and blocking with goat serum. The slides were then incubated with anti-AKTIP or anti-ERα antibody overnight at 4°C followed by biotin-conjugated secondary antibody (Dako, Carpinteria, CA) incubation at room temperature for 1 hr. 3,3′-diaminobenzidine (DAB, Amresco, Solon, OH) was used to detect signal from HRP. Histoscores on an arbitrary scale: 0, no immunoreactivity; 1, weak; 2, moderate; 3, intense; and 4, very intense were used to represent protein expression of AKTIP whereas histoscores of 0, no immunoreactivity; 1, weak; 2, moderate; 3, intense were used to represent protein expression of ERα. Our ERα staining scores are completely concordant with the reported ERα intensity provided by the company.
Biotin conjugated secondary antibody
Manufactured by Agilent Technologies
Sourced in United States, Denmark, United Kingdom
Biotin-conjugated secondary antibody is a lab equipment product that serves as a detection reagent. It is composed of a secondary antibody that has been conjugated with biotin, a small molecule that binds to the protein streptavidin. This product is commonly used in various immunoassay techniques to amplify and detect target proteins or antigens.
Automatically generated - may contain errors
Lab products found in correlation
3 3 diaminobenzidine dab, by Avantor (2 mentions)
Anti ki67, by Cell Signaling Technology (2 mentions)
Avidin biotin complex, by Vector Laboratories (2 mentions)
3 3 diaminobenzidine (dab), by Agilent Technologies (2 mentions)
3 3 diaminobenzidine (dab), by Avantor (1 mentions)
Horseradish peroxidase conjugated avidin, by Agilent Technologies (1 mentions)
Anti cd8, by Santa Cruz Biotechnology (1 mentions)
Entellan, by Merck Group (1 mentions)
Ab1316, by Abcam (1 mentions)
Anti cd3, by Agilent Technologies (1 mentions)
Tyramide amplification system, by PerkinElmer (1 mentions)
Cox 2, by Cayman Chemical (1 mentions)
Vectastain kit, by Vector Laboratories (1 mentions)
Tris edta buffer, by Agilent Technologies (1 mentions)
Bx51 microscope, by Olympus (1 mentions)
Xbp1s, by BioLegend (1 mentions)
Phospho eif2α, by Abcam (1 mentions)
Vimentin, by Agilent Technologies (1 mentions)
Cathepsin d, by Merck Group (1 mentions)
Uranyl acetate, by Agar Scientific (1 mentions)
12 protocols using biotin conjugated secondary antibody
1
Immunohistochemical Analysis of AKTIP and ERα
2
Immunohistochemical Analysis of AKTIP and ERα
3
Immunohistochemical Analysis of Ovarian Tumor
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Human ovarian tumor tissue array (OVC1021) was obtained from Pantomics (Richmond, CA). The slides were deparaffinized and rehydrated in graded ethanol prior to antigen retrieval using citrate buffer pH 6.0. They were then incubated with 3% H2O2 to quench endogenous peroxidase activity and goat serum for blocking. Incubation with primary antibody (1:25 for p85β and 1:40 for AXL) was performed at 4 °C overnight and subsequently with biotin-conjugated secondary antibody (Dako, Carpinteria, CA) for 1 h at room temperature. HRP was detected by DAB (Amresco, Solon, OH). Protein levels were represented by histoscore scores on an arbitrary scale: 0, no immunoreactivity; 1, weak; 2, moderate; 3, intense; and 4, very intense.
4
Immunohistochemical Analysis of Tumor Angiogenesis
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Formalin-fixed paraffin-embedded samples were sectioned into 4-μm slices. Deparaffinized sections were subjected to antigen retrieval with citrate solution, pH 6.0, and treated with 3% hydrogen peroxide to inhibit endogenous peroxidase activity. After blocking slides with FBS, sections were incubated overnight at 4°C with a 1:200 dilution of anti-vascular endothelial growth factor (VEGF) (Abcam, ab1316), 1:100 dilution of anti-VCAM-1 (Santa Cruz Biotechnology, sc-1504), anti-CD4 (Abcam, ab51312), and anti-CD8 (Santa Cruz Biotechnology, sc-7970) monoclonal antibody. Slides were treated with biotin-conjugated secondary antibody (Dako) and with horseradish peroxidase-conjugated avidin (Dako). Peroxidase activity was localized for all samples with 3,3′-diaminobenzidine, counterstained with hematoxylin, dehydrated, cleared, and mounted in mounting medium Entellan (Merck).
5
Immunofluorescent Detection of CCN1 in Arterial Sections
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Formalin-fixed, paraffin-embedded arterial sections were deparaffinised, rehydrated and subjected to antigen retrieval (sodium citrate buffer, 0.1 mol/L, pH = 6.0, at 100 °C for 10 min). Sections were blocked with Image-iT FX (Life Technologies) and incubated with 10 μg/ml sheep anti-CCN1 (R&D Systems, #AF4055), biotin-conjugated secondary antibody (Dako), followed by detection with Strepavidin Alexa-Fluor 488.
6
Immunofluorescent Analysis of p-Akt and p-mTOR in MDA-MB231 Cells
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MDA-MB231 cells (1 x 105/ml) cultivated in the absence or presence of Gαh/PLC-δ1 PPI inhibitor and grown on cover slides (22 mm in diameter and 0.17 mm in thickness) were fixed in 4% formaldehyde for 15 min at RT. After washing cells two times with PBS, the cells were treated with 95% EtOH/5% CH3COOH at-20°C for 15 min. Before blocking with 2% BSA/0.1% Triton X-100 for 2 hours at room temperature (RT), the cells were washed two times with PBS. Subsequently, the cells were incubated with p-Akt or p-mTOR antibody overnight at 4°C. After washing the cells three times with PBS, the cells were incubated with biotin-conjugated secondary antibody (DAKO) for 1 hour at RT. The cells were washed three times with PBS and incubated with fluorescein-conjugated avidin complex (Vector Laboratories) for 30 min at RT. After mounting the cells were analyzed using a FluoView confocal microscope system (Olympus).
7
Immunohistochemical Analysis of Tumor Samples
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Tumour and FL cells were fixed with 4% paraformaldehyde, embedded in paraffin and sectioned. Tissue sections were deparaffinized and rehydrated. For histology, slides were stained with hematoxylin and eosin (H&E). For immunohistochemistry, antigen retrieval was performed by heating the FL sections in 10 mM sodium citrate buffer (pH 6.0) in an electric pressure cooker, after which the slides were permeabilized with 0.05% Tween 20 in PBS. Blocking of endogenous peroxidase occurred in 3% H2O2 in methanol. Sections were then treated with 1% goat serum/1% BSA in PBS, followed by incubation with primary anti-RUNX2 rabbit monoclonal antibody (D1I7F; Cell signalling technology), anti-CD3 (Dako), anti-KI67 (Cell Signaling) overnight at 4 °C. Biotin-conjugated secondary antibodies (Dako) were detected by the avidin-biotin complex (Vector Laboratories, Burlingame, CA, USA), amplified with a tyramide amplification system (TSA, Perkin Elmer) and developed with diaminobenzidine (Dako).
8
Immunohistochemical Analysis of Tumor Samples
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Tumors were fixed with 4% paraformaldehyde, embedded in paraffin, and sectioned. Tissue sections were deparaffinized and rehydrated. For histology, slides were stained with H&E. For immunohistochemistry, antigen retrieval was performed by heating the sections in 10 mM sodium citrate buffer (pH 6.0) in an electric pressure cooker, after which the slides were permeabilized with 0.05% Tween 20 in PBS. Blocking of endogenous peroxidase occurred in 3% H2O2 in methanol. Sections were then treated with 1% goat serum/1% BSA in PBS, followed by incubation with primary anti-CD45R/B220 rat mAb (14-0452-85; eBioscience), anti-CD45 rat mAb (550539; BD Pharmingen), and anti-KI67 (Cell Signaling Technology) overnight at 4°C. Biotin-conjugated secondary antibodies (Dako) were detected by the avidin-biotin complex (Vector Laboratories), amplified with a tyramide amplification system (PerkinElmer), and developed with diaminobenzidine (Dako).
9
Immunohistochemistry for Inflammation Markers
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Frozen sections were processed for immunohistochemistry with antibodies against MPO (1:100; Dako, Carpinteria, CA, USA), iNOS (1:500, Transduction Laboratory) and COX-2 (1:200, Cayman Chemicals, Ann Arbor, MI, USA), as previously described [34 (link)]. The sections were incubated with primary antibodies, followed by biotin-conjugated secondary antibodies (Dako). The ABC method was used to detect labeled cells using a Vectastain kit (Vector Labs, Burlingame, CA, USA). Diaminobenzidine served as the substrate for peroxidase. Immunostaining control studies were performed by omission of the primary antibodies, by replacement primary antibodies with nonimmune, control antibody, and by preabsorption with an excess (10 µg/mL) of the respective antigens.
10
Immunohistochemical Detection of Embryonic Myosin
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The sections of paraffin-embedded muscle tissue were deparaffinized in xylene and rehydrated in ethyl alcohol. Then, the sections were blocked with 1% hydrogen peroxide (H2O2) in distilled water for 10 min, and the non-specific sites were blocked with bovine serum albumin (BSA, DAKO) for 20 min at room temperature. For detecting eMyHC, heat-induced antigen retrieval was performed (Tris/EDTA buffer, pH 8, DAKO) prior to staining the muscle samples. The sections were then incubated overnight at 4 °C with primary antibody of anti-eMyHC (clone BF-45, mouse, 1:400). The BF-45 monoclonal antibody was obtained from DSHB at the University of Iowa in USA. After thorough washing in PBS, the sections were incubated with biotin-conjugated secondary antibodies (DAKO) at 37 °C for 20 min. We used a standard peroxidase-based method with DAB (DAKO) to detect the antibody. The sections were dehydrated with ethyl alcohol and coverslipped with mounting medium. The stained sections were imaged using an Olympus BX51 microscope.
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