Biotinylated secondary antibody

Manufactured by Dianova

Sourced in Germany

Biotinylated secondary antibody is a lab equipment product used in various immunoassay techniques. It serves as a detection reagent, binding to the primary antibody and allowing for signal amplification through the interaction with streptavidin-enzyme conjugates.

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Lab products found in correlation

Metal enhanced diaminobenzidine, by Merck Group (2 mentions) Alkaline phosphatase kit, by Merck Group (2 mentions) Vectastain elite abc kit, by Vector Laboratories (2 mentions) Horseradish peroxidase conjugated streptavidin, by Vector Laboratories (1 mentions) Aquamount, by Polysciences (1 mentions) Hoechst, by Thermo Fisher Scientific (1 mentions) Streptavidin cy3, by Dianova (1 mentions) Streptavidin cy3, by Merck Group (1 mentions) Paraformaldehyde pbs, by Merck Group (1 mentions) Saponin, by Merck Group (1 mentions) Rabbit anti bmp4, by Abcam (1 mentions) Vectastain abc kit, by Vector Laboratories (1 mentions) Entellan, by Merck Group (1 mentions) Vectastatin elite abc kit, by Vector Laboratories (1 mentions) Hoechst 33342, by Thermo Fisher Scientific (1 mentions) Tsa cy3, by PerkinElmer (1 mentions) F4 80, by BD (1 mentions) Cy3 conjugated streptavidin, by Jackson ImmunoResearch (1 mentions) α sma, by Agilent Technologies (1 mentions) Ab5131, by Abcam (1 mentions)

Close spelling variants of this product

Biotinylated anti-rabbit secondary antibody (2 citations) Biotinylated goat anti-mouse secondary antibody (2 citations)

11 protocols using biotinylated secondary antibody

Histochemical and Immunohistochemical Analysis of Cartilage Aggregates

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Aggregates were harvested on d14 and d28, fixed in 4% paraformaldehyde and 10μm thick frozen sections were prepared.
Sections were stained with dimethylmethylene blue (DMMB) (Sigma Aldrich) for glycosaminoglycans. Histochemical ALP staining was performed with an alkaline phosphatase kit (Sigma Aldrich) with neutral red as counterstain.
For immunohistochemistry mouse anti collagen type X (1:20, Quartett Immunodiagnostika und Biotechnologie GmbH) and mouse anti collagen type II (1:100, Calbiochem) antibodies were used and immunohistochemistry was carried out as follows: . After blocking of endogenous peptidases (3% H₂O₂/ 10% Methanol in PBS) for 30 minutes, antigen retrieval with pepsin digestion for 15 minutes at room temperature (RT) was performed. Then sections were incubated in blocking buffer (10% fetal bovine serum/10% goat serum in PBS) for 60 minutes at RT followed by incubation in an appropriate primary antibody in blocking buffer overnight at 4°C. For collagen type X staining additional hyaluronidase digestion for 60 minutes at RT was performed prior to blocking. Immunolabeling was detected with a biotinylated secondary antibody (1:100; Dianova), horseradish peroxidase conjugated streptavidin (Vector Laboratories, Burlingame) and metal enhanced diaminobenzidine as substrate (Sigma Aldrich).

Pfeifer C.G., Karl A., Kerschbaum M., Berner A., Lang S., Schupfner R., Koch M., Angele P., Nerlich M, & Mueller M.B. (2019). TGF-β Signalling is Suppressed under Pro-Hypertrophic Conditions in MSC Chondrogenesis Due to TGF-β Receptor Downregulation. International Journal of Stem Cells, 12(1), 139-150.

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Immunohistochemistry of GLUT4 in Cryosections

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Cryosections for immunohistochemistry have been prepared as described previously^{12 (link)}. Sectioned samples were left 1 h at room temperature to dry, before being fixated in formaldehyde for 5 min. Sections were blocked in PBS (3% BSA) for 30 min, washed and subsequently incubated with GLUT4 (1:1000 in PBS (1% BSA), provided by A. Schürmann, DIfE Potsdam) for 1 h at room temperature. After washing, sections were incubated with a biotinylated secondary antibody (1:200 in PBS; Dianova, Germany) for 30 min, washed and incubated with Streptavidin-Cy3 (1:200 in PBS; Dianova, Germany) for 30 min. Subsequently the sections were washed and incubated with Hoechst (1:1000 in PBS; Thermo Fisher, Germany) for 5 min to stain for nuclear positioning, washed and mounted on slides using Aqua Mount (Polysciences, Germany).

Langer H.T., Afzal S., Kempa S, & Spuler S. (2020). Nerve damage induced skeletal muscle atrophy is associated with increased accumulation of intramuscular glucose and polyol pathway intermediates. Scientific Reports, 10, 1908.

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Immunolabeling of Insect Neuron Epitopes

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All three groups of embryos were processed for immunofluorescence labeling. Embryos were fixed in 4% paraformaldehyde/PBS (Sigma) for 60 minutes, followed by permeabilization with 0.3% saponin (Sigma) in PBS with Triton-X100 (0.1%, Sigma, PBS-T) for 45 minutes. Then, the embryos were washed in PBS-T 0.1% for 3 times before they were blocked (30 minutes) with PBS-T 0.1% containing 5% normal rabbit serum (NRS, Linaris).
The Ti1-pioneer neurons were labeled with HRP antibodies that are able to detect a specific carbohydrate epitope expressed on the neuronal surface of insect neurons^{24 (link),25 (link)}. The antibody (goat-anti-HRP, Dianova) was diluted 1:2000 in blocking solution, containing PBS-T 0.1% and 5% NRS (Linaris). Next, the embryos were incubated with HRP antibodies over night at 4 °C.
Following the immunolabeling, preparations were washed three times for 10 minutes respectively, before they were incubated with the biotinylated secondary antibody (1:250, rabbit-anti-goat in blocking solution, Dianova) for one hour at room temperature (RT). After three additional washing steps for 10 minutes in PBS-T, the second antibody was detected by incubation for one hour at RT with Streptavidin-Cy3 (Sigma), diluted in PBS-T 0.1% (1:250).

Bode K., Nolte L., Kamin H., Desens M., Ulmann A., Bergmann G.A., Betker P., Reitmeier J., Ripken T., Stern M., Meyer H, & Bicker G. (2020). Scanning laser optical tomography resolves developmental neurotoxic effects on pioneer neurons. Scientific Reports, 10, 2641.

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Histochemical and Immunohistochemical Analysis

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For histochemical investigations 10 μm thick sections were prepared as well. Histochemical ALP staining was performed with an alkaline phosphatase kit (Sigma Aldrich) with neutral red as counterstain.
For immunohistochemistry mouse anti-BAMBI (1 : 10, eBioscience), rabbit anti-BMP4 (1 : 250, Abcam), mouse anti-collagen type X (1 : 20, Quartett Immunodiagnostika und Biotechnologie GmbH), and mouse anti-collagen type II (1 : 100, Calbiochem) antibodies were used and immunohistochemistry was carried out as follows: after rinsing samples in washing buffer for 5 minutes blocking of endogenous peptidases (3% H₂O₂/10% methanol in PBS) was performed for 30 minutes. Then sections were incubated in blocking buffer (10% fetal bovine serum/10% goat serum in PBS) for 60 minutes at RT followed by incubation anti-BAMBI primary antibody in blocking buffer overnight at 4°C. Immunolabeling was detected by a biotinylated secondary antibody (1 : 100; Dianova), horse reddish peroxidase conjugated streptavidin (Vector Laboratories, Burlingame), and metal enhanced diaminobenzidine as a substrate (Sigma Aldrich).

Pfeifer C.G., Karl A., Berner A., Zellner J., Schmitz P., Loibl M., Koch M., Angele P., Nerlich M, & Mueller M.B. (2016). Expression of BMP and Actin Membrane Bound Inhibitor Is Increased during Terminal Differentiation of MSCs. Stem Cells International, 2016, 2685147.

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Immunohistochemistry of Human Ovarian DDC

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Sections of human ovaries derived from a local collection at Anatomy III, Cell Biology (Munich, Germany) were used for immunohistochemistry. Immunohistochemistry was performed with a rabbit antiserum raised against human DDC (Sigma-Aldrich) and the corresponding blocking peptide (Sigma-Aldrich). Tissue samples and immunohistochemistry were described previously [5 (link), 18 (link)]. In brief, after removal of paraffin, antigen retrieval and blocking of endogenous peroxidase activity, the tissue was incubated in 5 % appropriate serum, diluted in phosphate-buffered saline. Antiserum incubation was done overnight at 4 °C. The antiserum against human DDC was diluted 1:50. Afterwards, incubation with a biotinylated secondary antibody (1:500 dilution; Dianova, Hamburg, Germany) for 2 h at room temperature was performed. A Vectastain ABC Kit (Vector Laboratories, Burlingame, CA, USA) and a 3,3′-diaminobenzidine tablet set (Sigma-Aldrich) were used for the final staining procedure. Slides were covered with Entellan (Merck Millipore, Billerica, MA, USA). For control purposes, incubation with antigen peptide pre-adsorbed antiserum was employed.

Blohberger J., Buck T., Berg D., Berg U., Kunz L, & Mayerhofer A. (2016). L-DOPA in the hu man ovarian follicular fluid acts as an antioxidant factor on granulosa cells. Journal of Ovarian Research, 9, 62.

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Iba1 Immunohistochemistry for Microglia

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Immunohistochemistry for the Iba1 antigen as a marker for microglia and macrophages was performed following a well-established staining protocol [28] (link). Briefly, free-floating 1-in-12 section series were treated with 0.6% H₂O₂ to deactivate endogenous tissue peroxidases. After 30 min background blocking with PBS enriched with 3% donkey serum (PBS+), sections were incubated with primary anti-Iba1 (rabbit, 1∶1000, Wako) antibody overnight at 4°C. The next day, after washing with PBS and blocking with PBS+, sections were incubated with biotinylated secondary antibody (anti-rabbit, 1∶250, dianova) for 2 h at room temperature (RT). ABC reagent (Vectastatin ABC Elite Kit, Vector Laboratories) was applied for 1 h. Finally, sections were incubated with Diaminobenzidine (DAB)/peroxidase (Sigma, Germany) in a solution containing 0.3% H₂O₂ and 0.01% nickel chloride for at least 5 min at RT. Sections were mounted on microscope slides and coverslipped for later quantification.

Klein C., Hain E.G., Braun J., Riek K., Mueller S., Steiner B, & Sack I. (2014). Enhanced Adult Neurogenesis Increases Brain Stiffness: In Vivo Magnetic Resonance Elastography in a Mouse Model of Dopamine Depletion. PLoS ONE, 9(3), e92582.

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Immunohistochemical Analysis of Skin Cryosections

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Immunohistochemistry of 8μm skin cryosections was performed using the fluorescence microscope Olympus IX81 (Olympus, Tokyo, Japan) and the TSA Cy3 (PerkinElmer, Waltham, MA) as recommended by the company. The following primary antibodies were used: F4/80 (BD Pharmingen), myeloperoxidase (Abcam, Cambridge, MA). The slides were incubated for 30 minutes at room temperature with the biotinylated secondary antibody (Dianova, Hamburg, Germany; BD). Nuclei were counterstained with Hoechst 33342 (Invitrogen). Tissues were mounted in Vectashield H-1000.

Klebow S., Hahn M., Nikoalev A., Wunderlich F.T., Hövelmeyer N., Karbach S.H, & Waisman A. (2016). IL-6 Signaling in Myelomonocytic Cells Is Not Crucial for the Development of IMQ-Induced Psoriasis. PLoS ONE, 11(3), e0151913.

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Immunofluorescence Staining of Murine Cardiac Fibroblasts

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Murine cardiac fibroblasts were incubated with external stimuli for up to 72 h. After washing once with PBS (PAA), cells were fixed with 4% paraformaldehyde for 10 min. Cells were permeabilized with Triton X-100 and incubated with avidin blocking solution for 30 min. Then, cells were treated with the primary antibody α-SMA (1:50; Dako, Hamburg, Germany) in the presence of biotin solution for 90 min. Afterwards, fibroblasts were washed twice followed by 1 h incubation with the biotinylated secondary antibody (1:250; Dianova, Hamburg, Germany). As described previously^{11 (link)}, cells were finally incubated 30 min with Cy3-conjugated streptavidin (1:250; Jackson ImmunoResearch Laboratories Inc., West Grove, PA, USA) for fluorescence visualization and with Diamidinphenylindol (DAPI; 1:100; Invitrogen, Darmstadt, Germany) as nuclear staining. All incubations were performed at room temperature.

Pappritz K., Savvatis K., Koschel A., Miteva K., Tschöpe C, & Van Linthout S. (2018). Cardiac (myo)fibroblasts modulate the migration of monocyte subsets. Scientific Reports, 8, 5575.

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Immunohistochemical Analysis of Rat Testes

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Sections (4–5 µm) from rat testes were immunohistochemically analyzed according to standard protocols (Bergmann and Kliesch, 1994 (link); Kliesch et al., 1998 (link)) with minor modifications. Sections were incubated with primary antibody overnight at 4°C, and then for 1 h at room temperature with biotinylated secondary antibody (Dianova; 1:250), followed by incubation with avidin–biotin complex (Vectastain ABC Elite Kit, Vector Labs, Burlingame, CA, USA) for 45 min with 3,3′-diaminobenzidine as chromogen. The primary antibodies used were: rabbit polyclonal anti-trimethyl-H3K79 (ab2621; Abcam; 1:1000); rabbit polyclonal anti-acetyl-histone H4 antibody (Millipore 06-598; 1:500); or rabbit polyclonal to active RNA polymerase II (CTD repeat YSPTSPS, phospho S5; ab5131; Abcam; 1:500). Sections were analyzed using a Zeiss Axioplan light microscope equipped with a Zeiss AxioCam MR^m digital camera.
This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the German Animal Welfare Act and local regulations at JLU Giessen.

Dottermusch-Heidel C., Gärtner S.M., Tegeder I., Rathke C., Barckmann B., Bartkuhn M., Bhushan S., Steger K., Meinhardt A, & Renkawitz-Pohl R. (2014). H3K79 methylation: a new conserved mark that accompanies H4 hyperacetylation prior to histone-to-protamine transition in Drosophila and rat. Biology Open, 3(6), 444-452.

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Murine Femoral Fracture Histology

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Murine femoral fracture sections were used for histological analysis. After standard processing, serial transverse 5-µm sections were cut and stained with hematoxylin/eosin for histological assessment. For immunohistochemistry with an antiserum against TFF3 [34 (link)], deparaffinized sections were pretreated with 0.05% trypsin for 60 min. The sections were then left to react with anti-human TFF3, serum (anti-rTFF3-1) [34 (link)] diluted 1:300, followed by incubation with biotinylated secondary antibodies (Dianova, Hamburg, Germany). Negative control sections were incubated with isotype normal mouse IgG (Santa Cruz Biotechnology). Bound antibodies were visualized by exposure to a complex of avidin and biotinylated peroxidase H (Vectastain-Elite-ABC Kit, Vector Laboratories, Burlingame, CA). The sections were developed with AEC (3-amino-9-ethylcarbazole) (DAKO, Hamburg, Germany) and counterstained with hematoxylin.

Krüger K., Schmid S., Paulsen F., Ignatius A., Klinger P., Hotfiel T., Swoboda B, & Gelse K. (2019). Trefoil Factor 3 (TFF3) Is Involved in Cell Migration for Skeletal Repair. International Journal of Molecular Sciences, 20(17), 4277.

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