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Biotinylated goat anti rabbit secondary antibody

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Biotinylated goat anti-rabbit secondary antibody is a reagent used in various immunoassays and detection methods. It is produced by immunizing goats with rabbit IgG and labeling the resulting antibodies with biotin. This secondary antibody can be used to detect the presence of rabbit primary antibodies in samples.

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Lab products found in correlation

Abc kit, by Vector Laboratories (4 mentions) 4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (3 mentions) Permount, by Merck Group (2 mentions) Rabbit anti fos, by Santa Cruz Biotechnology (2 mentions) Histoclear, by Merck Group (2 mentions) Avidin biotin complex kit, by Vector Laboratories (2 mentions) Rotenone, by Merck Group (2 mentions) Anti tyrosine hydrolase polyclonal rabbit antibody, by Merck Group (2 mentions) Rabbit anti c fos primary antibody, by Merck Group (2 mentions) Ucp1 antibody, by Abcam (2 mentions) Cy3 streptavidin, by Jackson ImmunoResearch (2 mentions) Diaminobenzidine substrate, by Vector Laboratories (2 mentions) Monoclonal mouse anti α synuclein, by BD (2 mentions) Alexa flour 488 conjugated goat anti rabbit secondary antibodies, by Thermo Fisher Scientific (2 mentions) Anti ionized calcium binding adaptor molecule 1 iba 1, by Fujifilm (2 mentions) Anti glial fibrillary acidic protein, by Merck Group (2 mentions) Panoramic viewer, by 3DHISTECH (1 mentions) Tsa cy3 system, by PerkinElmer (1 mentions) Tcs sp5 2 confocal microscope, by Leica (1 mentions) Microtome, by Leica (1 mentions)

Spelling variants of this product

Biotinylated secondary antibody (73 citations) Anti-rabbit secondary antibody (44 citations) Goat anti-rabbit secondary antibody (43 citations) Biotinylated goat anti-rabbit (14 citations) Biotinylated anti-rabbit secondary antibody (8 citations) Biotinylated goat anti-rabbit antibody (4 citations) Biotinylated anti-goat secondary antibody (3 citations) Goat-anti-rabbit IgG-biotinylated secondary antibody (2 citations)

24 protocols using biotinylated goat anti rabbit secondary antibody

1

Analyzing Human Liver Allograft Tissue

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Analysis of human liver allograft tissue (PRO10110393) and isolation of human primary hepatocytes (PRO012100076 and PRO08010372) were conducted under University of Pittsburgh Institutional Review Board protocols. Written informed consent was received from participants prior to inclusion in the study. Formalin-fixed, paraffin-embedded human liver allograft biopsy sections were obtained from 4 patients at two different time points (backtable and post-reperfusion (1–4 h)). 4 μm sections were deparaffinized, hydrated, and treated with citrated buffer for antigen retrieval. Sections were then blocked with avidin and biotin block kit (Vector Laboratories, Inc., Burlingame, CA). Staining was performed by sequential incubation cycles of rabbit anti-IRF-1 primary antibody (Santa Cruz Biotechnology, Inc., Dallas, TX), goat anti-rabbit biotinylated secondary antibody (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA), ABC kit (Vector Laboratories, Inc.), and AEC Substrate kit (ScyTek Laboratories, Inc., Logan, UT). Sections were then counterstained with aqueous hematoxylin. Digital images of whole staining slides were obtained with MIRAX MIDI digital whole slide scanning system (Carl Zeiss Microimaging, Jena, Germany) and analyzed with Panoramic Viewer (3D Histech, Ltd, Ramsey, NJ).
Yokota S., Yoshida O., Dou L., Spadaro A.V., Isse K., Ross M.A., Stolz D.B., Kimura S., Du Q., Demetris A.J., Thomson A.W, & Geller D.A. (2015). IRF-1 promotes liver transplant ischemia/reperfusion injury via hepatocyte IL-15/IL-15Rα production. Journal of immunology (Baltimore, Md. : 1950), 194(12), 6045-6056.
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2

Immunohistochemical Analysis of C. rodentium Infection

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For immunohistochemical (IHC) stainings, distal colonic tissues from mice infected with C. rodentium were fixed in buffered formalin (Roti-Histofix; Carl Roth) at 4 °C for 24 h, dehydrated, and embedded in liquid paraffin. 3-µm sections were cut using a microtome (Leica) and processed for IHC applying the Tyramide Signal Amplification (TSA) Cy3 system (Perkin Elmer) according to the manufacturer’s protocol. To analyze the expression level of IRF-1, a primary antibody from Cell Signaling (D5E4) was used (1:50 dilution). To visualize the colonization of the mucosal surface with C. rodentium, a primary antibody from Abcam (ab37056, 1:1000) was applied. Both primary antibodies were used in combination with a goat-anti-rabbit biotinylated secondary antibody (Jackson Immuno Research). Epithelial cells were stained with Alexa Fluor 488 anti-mouse CD326 (Ep-CAM; G8.8; BioLegend, 1:100). Nuclei were counterstained with DAPI (Invitrogen). Pictures were acquired on a Leica TCS SP5 II confocal microscope using Leica LAS AF version 2.7.3.9723 software.
Schmalzl A., Leupold T., Kreiss L., Waldner M., Schürmann S., Neurath M.F., Becker C, & Wirtz S. (2022). Interferon regulatory factor 1 (IRF-1) promotes intestinal group 3 innate lymphoid responses during Citrobacter rodentium infection. Nature Communications, 13, 5730.
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3

Immunolabeling Protocol for Tissue Sections

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After resectioning, 50 µm sections were washed in PBS for at least 10 min prior to receiving 0.1M glycine made in 0.05M PBS for 30 min. The tissue was subsequently washed three times in PBS for 5 min each wash. Next, sections received 0.5% sodium borohydride in PBS for 15 min. Sections were then washed twice for 5 min in PBS before blocking in 5% NGS, 0.5% Tx, and 1% H2O2 in PBS for 30 min. After blocking, sections received one of three primary antisera for two days: a monoclonal anti‐peripherin (1:300; Chemicon International, Temecula, CA), a polyclonal anti‐VIP (1:8,000; Immunostar, Inc. Hudson, WI), or a polyclonal anti‐MUC2 (3 µg/ml; Novus Biologicals). After primary sections were washed with 1% NGS in PBS four times for 15 min each wash. Next, secondary antibody was added for 2 hr at room temperature and consisted of 1% NGS and 0.5% Tx in PBS with a biotinylated goat anti‐rabbit secondary antibody (1:2,500; Jackson Immunoresearch Inc. West Grove, PA). Secondary antibody was washed out with four 15 min washes composed of 0.02% Tx in PBS. Sections were next incubated with an Alexa Fluor 488 conjugated to streptavidin (1:500; Invitrogen) in 0.32% Tx in PBS for 1 hr. Finally, sections received three PBS washes prior to mounting and imaging.
Schwerdtfeger L.A, & Tobet S.A. (2020). Vasoactive intestinal peptide regulates ileal goblet cell production in mice. Physiological Reports, 8(3), e14363.
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4

Quantifying Microglial Response in Stroke

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In order to examine changes in microglia, two sections were chosen between 0.7mm anterior and 0.3 mm posterior to bregma for analysis. The sections were washed with 0.1M phosphate buffered saline (PBS) before being incubated in 40% methanol 2% H2O2 in PBS for 20 min at room temperature. The sections were washed again and blocked with 10% normal goat serum with 0.03% Triton ×100 in PBS for 1 hour. After blocking, sections were incubated in rabbit anti-Iba-1, which labels ionized calcium binding adaptor molecule 1 present in myeloid cells (1:1000, Wako, cat. #011974) overnight at 4°C. The following day, the sections were washed and then incubated in biotinylated goat anti-rabbit secondary antibody (1:300, Jackson ImmunoResearch) for 1 hour at room temperature. After washing, the sections were incubated in ABC solution (Vector Laboratories) for 1 hour, washed again and then the immunolabeling visualized using DAB (Pierce). The slides were then dried, dehydrated through a graded series of alcohols, cleared in xylene and coverslipped with DPX. The sections were examined on an Olympus BX60 microscope at 20×, the view field aligned to medial edge of the infarct and the image captured with a Magnafire digital camera. The number of IBa1 labeled cells was counted using a semi-automated process with Image-Pro Plus software (Media Cybernetics).
Womble T.A., Green S., Shahaduzzaman M., Grieco J., Sanberg P.R., Pennypacker K.R, & Willing A.E. (2014). Monocytes are Essential for the Neuroprotective Effect of Human Cord Blood Cells Following Middle Cerebral Artery Occlusion in Rat. Molecular and cellular neurosciences, 59, 76-84.
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5

Immunohistochemical Visualization of c-Fos Expression

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Free-floating sections were first washed 3 times in 0.01 M PBS and between each subsequent step except as noted. Sections were incubated in 1% hydrogen peroxide, followed by 5% normal goat serum (NGS) and 0.25% Triton X in PBS. Sections were incubated for 48 h at 4 °C in primary antibody directed against c-Fos (rabbit anti-Fos, 1:3,000, Santa Cruz Biotechnology) in PBS with 5% NGS and 0.25% Triton X. Sections were then incubated in biotinylated goat anti-rabbit secondary antibody (1:200, Jackson Labs) for 2 h, followed by incubation in avidin biotin complex (ABC kit, Vector Laboratories) for 2 h. Sections were washed 3 times in 0.1 M PB, then immunoreactivity was visualized with SG (SG substrate kit, Vector Laboratories). Sections were mounted on slides using a 0.0015% gelatin solution, dehydrated using a series of ethanol solution, defatted using Histoclear (Sigma-Aldrich), and coverslipped with Permount (Sigma-Aldrich).
Fontenot J., Loetz E., Ishiki M, & Bland S.T. (2017). Monoacylglycerol lipase inhibition alters social behavior in male and female rats after post-weaning social isolation. Behavioural brain research, 341, 146-153.
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6

Immunofluorescent Visualization of ERα

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Brain sections were prepared as same as in immunoperoxidase labelling. After thorough washing in PBS, sections were incubated for 1 hr with blocking solution (PBS containing 3% normal goat serum and 0.25% Triton-X), and then incubated overnight in blocking solution containing rabbit anti-ERα antibody (1:1000, Millipore, Billerica, MA). After washing in PBS, sections were incubated in blocking solution containing biotinylated goat anti-rabbit secondary antibody (1:1000, Jackson Immunoresearch Laboratories, Inc) for 2 hours, then washed again and incubated in blocking solution containing Dylight 594 streptavidin (1:500, Jackson Immunoresearch Laboratories, Inc.). After washing in PBS, the sections were mounted on Poly-L-Lysine glass slides and air dried. Finally, they were coverslipped with Vectashield mounting medium with DAPI (Vector Laboratories). Under the Leica 5500 fluorescence microscope equipped with optigrid illumination, native ZsGreen signals were directly visualized with the green fluorescence filter and ERα immunoreactivity was visualized with the red fluorescence filter.
Saito K., He Y., Yan X., Yang Y., Wang C., Xu P., Hinton AO J.r., Shu G., Yu L., Tong Q, & Xu Y. (2015). Visualizing estrogen receptor-α-expressing neurons using a new ERα-ZsGreen reporter mouse line. Metabolism: clinical and experimental, 65(4), 522-532.
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7

Immunohistochemistry Analysis of Fos Expression

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Immunohistochemistry was performed for Fos as previously reported (wall). Free-floating sections were first washed 3 times in 0.01 M PBS and between each subsequent step except as noted. Sections were incubated in 0.3% hydrogen peroxide, followed by 5% normal goat serum (NGS) and 0.25% Triton X in PBS. Sections were incubated overnight at RT in rabbit anti-Fos, (1:10,000, Santa Cruz Biotechnology) in PBS with 5% NGS and 0.25% Triton X. Sections were then incubated in biotinylated goat anti-rabbit secondary antibody (1:200, Jackson Labs) for 2 h, followed by incubation in avidin biotin complex (ABC kit, Vector Laboratories) for 2 h. Sections were washed 3 times in 0.1 M PB, then immunoreactivity was visualized with 3,3’-diaminobenzidine (DAB substrate kit, Vector Laboratories) and nickel ammonium sulphate as chromogens. Sections were mounted on slides using a 0.15% gelatin solution, dehydrated using a series of ethanol solutions, defatted using Histoclear (Sigma-Aldrich), and coverslipped with Permount (Sigma-Aldrich). Representative photomicrographs from each of the regions of interest are shown in Figure 5.
Ahern M., Goodell D.J., Adams J, & Bland S.T. (2015). Brain regional differences in social encounter-induced Fos expression in male and female rats after post-weaning social isolation. Brain research, 1630, 120-133.
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8

Microglia and Astrocyte Immunohistochemistry

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Brains were sectioned at 40 μm (Microm HM 505e cryostat). Every 6th section through the dorsal hippocampus/lateral hypothalamus was evaluated for glial fibrillary acid protein (GFAP, 1:1,000, Abcam, Cambridge, MA, USA) and ionized calcium binding adaptor protein (Iba-1, 1:1,000, FUJIFILM Wako, Osaka, Japan) immunoreactivity. Following primary antibody incubation, tissue was washed in PBST and then incubated in biotinylated goat anti-rabbit secondary antibody (1:500, Jackson ImmunoResearch, West Grove, PA, USA) for 2 hours. The tissue was washed in PBST and then incubated in ABC reagent (Vector Laboratories, Burlingame, CA, USA) for 1.5 hours. The tissue was developed with a Vector VIP kit (Vector Laboratories, Burlingame, CA, USA) and analyzed using a light microscope.
Daly C.M., Saxena J., Singh J., Bullard M.R., Bondy E.O., Saxena A., Buffalino R.E., Melville M.F, & Freeman L.R. (2020). Sex Differences in Response to a High Fat, High Sucrose Diet in both the Gut Microbiome and Hypothalamic Astrocytes and Microglia. Nutritional neuroscience, 25(2), 321-335.
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9

Molecular Mechanisms in Neuroinflammation

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Rotenone, Valeric acid, RIPA lysis buffer, antibodies against inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (Cox-2) and glial fibrillary acidic protein (GFAP) were procured from Sigma-Aldrich, St. Louis, MO, USA. Protease and phosphatase inhibitor cocktail were procured from Thermo Scientific, USA. Anti-tyrosine hydrolase (Polyclonal rabbit) antibody was obtained from Merck, Germany. The following antibodies were purchased from Cell Signalling Technology, Beverly, MA, USA: LC3, p62, mTOR, phosphor mTOR and p70S6K. Apoptotic polyclonal markers (Bax and Bcl-2) were obtained from Abcam, USA. Monoclonal mouse anti-α-synuclein antibody was purchased from BD Biosciences, San Jose, CA, USA. Anti- Iba-1 antibody was purchased from Wako chemicals, Richmond, VA, USA. Fluorescent secondary antibodies (Alexa Flour 488) were purchased from Thermo Fischer Scientific, Waltham, MA, USA. Biotinylated goat anti-rabbit secondary antibody was purchased from Jackson Immunoresearch, West grove, PA, USA. Biochemical assays were performed using commercially available kits. All other chemicals used in this experiments were provided by local commercial sources (analytical grade quality).
Jayaraj R.L., Beiram R., Azimullah S., MF N.M., Ojha S.K., Adem A, & Jalal F.Y. (2020). Valeric Acid Protects Dopaminergic Neurons by Suppressing Oxidative Stress, Neuroinflammation and Modulating Autophagy Pathways. International Journal of Molecular Sciences, 21(20), 7670.
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10

Quantification of Fos-Expressing Neurons

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All rats were overdosed with sodium pentobarbital and transcardially perfused. Brains were dissected and stored in 10% formalin for up to 14 h and then transferred to 30% sucrose at 4 °C for at least 72 h. After all brains were sectioned on a cryostat (−20°) at 30 μm and brain sections. For Fos immunohistochemistry, brain sections were washed 3x in TBST, incubated in 0.3% H20h for 15 min, washed 3x in TBS, and then incubated in rabbit anti-c-fos primary antibody (1:1000; Millipore) overnight. The next day the sections were washed 3x in TBS followed by a 1-h incubation in a biotinylated goat anti-rabbit secondary antibody (1:1000, Jackson Immunoresearch), amplified with the avidin biotin complex at 1:1000 (ABC; Vector labs), and visualized with 3,3’-diaminobenzidine (DAB) + nickel ammonium sulfate. Stained sections were mounted in subbed slides, coverslipped with Permount and stored at room temperature until they were imaged.
To quantify c-Fos expression, one 10X image (895 μm × 670 μm; 0.596 mm2) was taken of each hemisphere at different AP levels of each the PL and IL (+2.7 mm and +2.3 mm from bregma), HPC (−5.5 mm and −6.0 mm), and MGN (−5.5 mm and −6.0 mm). The total number of Fos-expression neurons in each image was manually counted, averaged across all images for each image, and divided by the surface area to the average number of Fos cells/mm2.
Totty M.S., Tuna T., Ramanathan K.R., Jin J., Peters S.E, & Maren S. (2023). Thalamic nucleus reuniens coordinates prefrontal-hippocampal synchrony to suppress extinguished fear. Nature Communications, 14, 6565.
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