Avidin biotin complex system

Manufactured by Vector Laboratories

Sourced in United States

The Avidin-biotin complex system is a highly versatile and sensitive tool used in various molecular and cellular biology applications. It is based on the strong non-covalent interaction between the protein avidin and the small molecule biotin. This system allows for the detection, purification, and immobilization of biomolecules labeled with biotin.

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Lab products found in correlation

Lc814, by Tissue Array (2 mentions) Target retrieval solution, by Agilent Technologies (2 mentions) Pk 6101, by Vector Laboratories (2 mentions) 3 30 diaminobenzidine tetrahydrochloride, by Merck Group (2 mentions) Abc elite kit, by Vector Laboratories (2 mentions) Fascin, by Santa Cruz Biotechnology (1 mentions) Diaminobenzidine kit, by Vector Laboratories (1 mentions) 3 3 diaminobenzidine (dab), by Vector Laboratories (1 mentions) Abc kit, by Vector Laboratories (1 mentions) Rat anti f4 80, by Bio-Rad (1 mentions) Anti ki67, by Abcam (1 mentions) Cd11b, by BD (1 mentions) Troma 1, by Developmental Studies Hybridoma Bank (1 mentions) Mouse anti tyrosine hydroxylase, by Merck Group (1 mentions) Biotinylated goat anti mouse igg, by Jackson ImmunoResearch (1 mentions) Mouse anti parvalbumin pv, by Merck Group (1 mentions) Stereoinvestigator system, by MicroBrightField (1 mentions) Anti nf κb p65, by Santa Cruz Biotechnology (1 mentions) Anti annexin 5, by Thermo Fisher Scientific (1 mentions) Anti stat3, by Santa Cruz Biotechnology (1 mentions)

16 protocols using avidin biotin complex system

Immunohistochemical Staining and Scoring Protocol

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Detailed procedures for the immunohistochemical assays and scoring system are described elsewhere [36 (link), 37 (link)]. Briefly, each tissue slide was reacted with monoclonal antibodies against Slug (Cell Signaling Technology); fascin (Santa Cruz Biotechnology); or CLDN-1, -2, or -3 (Novus Biologicals) in a humidified chamber for 60 min at 37°C. Afterwards, the sections were washed with PBS and treated with an appropriate biotin-conjugated secondary antibody for 30 min. The signal for each tissue section was detected using the avidin-biotin complex system and diaminobenzidine kit (Vector Laboratories, Burlingame, CA, USA). Immunohistochemical staining was quantified according to the coverage area and intensity for Slug as described [38 (link)]. The scoring system for the CLDNs and fascin is described elsewhere [39 (link)], with the following modifications: –, no staining or membrane staining in < 10% of tumor cells; +, faint, weakly perceptible positive staining of the membrane in < 10% of tumor cells; ++, weak-to-moderate complete membrane staining observed in ≥ 10% of tumor cells; and +++, strong, complete membrane staining in ≥ 10% of tumor cells.

Chang C.W., Yu J.C., Hsieh Y.H., Yao C.C., Chao J.I., Chen P.M., Hsieh H.Y., Hsiung C.N., Chu H.W., Shen C.Y, & Cheng C.W. (2016). MicroRNA-30a increases tight junction protein expression to suppress the epithelial-mesenchymal transition and metastasis by targeting Slug in breast cancer. Oncotarget, 7(13), 16462-16478.

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Immunohistochemical Analysis of RSK2 and Stathmin in Lung Cancer Metastasis

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Approval of use of human specimens was given by the Emory University Institutional Review Board. All of the clinical samples were collected with informed consent under Health Insurance Portability and Accountability Act approved protocols.
Paraffin-embedded lung cancer with matched lymph node metastasis tissue array (LC814) was obtained from US Biomax, Inc. (Rockville, MD, USA). IHC analysis of RSK2 expression and stathmin phosphorylation was performed using tissue array samples as previously described.^{46 (link)} In brief, human tissue sections were incubated in 3% hydrogen peroxide after deparaffinization and rehydration. Antigen retrieval was achieved by microwaving the sections in 100 mM Tris (pH 10.0) and 10 mM sodium citrate (pH 6.0) for RSK2 and phospho-stathmin S16 staining, respectively. The slides were subsequently blocked with 2.5% horse serum and avidin–biotin complex system (Vector Laboratories, Burlingame, CA, USA). The primary antibodies, anti-RSK2 antibody and anti-phospho-stathmin S16 antibody, were applied at a dilution of 1:100. Detection was achieved with 3,3′-diaminobenzidine and counterstained with hematoxylin. IHC staining results were scored as 0 for no staining, 1+ for weak staining, 2+ for moderate staining and 3+ for strong staining.

Alesi G., Jin L., Li D., Magliocca K., Kang Y., Chen Z., Shin D., Khuri F, & Kang S. (2016). RSK2 signals through stathmin to promote microtubule dynamics and tumor metastasis. Oncogene, 35(41), 5412-5421.

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Peptide Effects on Brain Histology

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Female BALB/C mice were randomly divided into three experimental groups with 10 mice in each group. Group A mice were used as a blank control; group B mice were injected with 5 μl of physiological saline containing 0.25 mg of non-specific peptide per mouse; group C mice were injected with 5 μl of physiologic saline containing 0.25 mg of the synthetic mimic peptide per mouse. After injection, all mice were monitored and housed in the animal facilities. Brain specimens were obtained at the specified time points and processed for histological examination. Paraffin-embedded sections (4 mm) were stained with HE. Paraffin-embedded sections (4 mm) were deparaffinized through graded ethanol solutions. After an antigen retrieval procedure of 30 min using target retrieval solution (DAKO), the sections were stained with the antibody of cleaved caspase-3 (Cell Signaling Technology) using the avidin-biotin complex system (Vector Laboratory). DAB was used as the substrate. Cell nuclei were counterstained with haematoxylin.

Teng Y., Liu S., Guo X., Liu S., Jin Y., He T., Bi D., Zhang P., Lin B., An X., Feng D., Mi Z, & Tong Y. (2017). An Integrative Analysis Reveals a Central Role of P53 Activation via MDM2 in Zika Virus Infection Induced Cell Death. Frontiers in Cellular and Infection Microbiology, 7, 327.

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Histological Analysis of Mouse Liver

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Mouse livers were fixed in 10% neutral-buffered formalin for 24 hours at room temperature and processed for paraffin embedding according to standard protocols. For frozen sections, livers were directly embedded in optimum cutting temperature compound and stored at −80°C. The tissue sections were deparaffinized and rehydrated through dimethylbenzene and graded ethanol. Tissue sections were stained with H&E using standard protocols. For IHC analysis, sections were boiled for 40 min in retrieval solution or treated with proteinase K (20 μg/ml) for antigen retrieval. Subsequently, sections were stained with specific antibodies using the avidin-biotin complex system (Vector Laboratories). Table S8 details the antibodies used for IHC. Signal was detected using the ABC kit, and 3,3′-diaminobenzidine (DAB) (Vector Laboratories) was used as the substrate for color development. Sections were counterstained with hematoxylin solution. Sirius red and oil red stainings were performed using respective kits (purchased from Servicebio) following the manufacturers’ instructions.

Tang M., Zhao Y., Zhao J., Wei S., Liu M., Zheng N., Geng D., Han S., Zhang Y., Zhong G., Li S., Zhang X., Wang C., Yan H., Cao X., Li L., Bai X., Ji J., Feng X.H., Qin J., Liang T, & Zhao B. (2022). Liver cancer heterogeneity modeled by in situ genome editing of hepatocytes. Science Advances, 8(25), eabn5683.

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Visualizing Nuclear c-Fos Expression via DAB Staining

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We used DAB staining to better visualize nuclear cfos expression over a relatively large area. 30 minutes after i.p. dexmedetomidine (50, 100, 400 μg/kg), or saline (0.9%) administration, experimental mice were anesthetized, perfused transcardially, and free-floating sections processed for immunohistochemistry with rabbit antibody to Fos^{29 (link)} (1:20,000, Ab-5, Calbiochem) after blocking endogenous peroxidase with 0.3% H₂O₂ in PBS.The primary antiserum was localized using a variation of the avidin-biotin complex system (Vector Laboratories). In brief, sections were incubated for 120 minutes at 22–25 °C in a solution of biotinylated goat antibody to rabbit IgG (PK-6101, Vector Laboratories) and then placed in the mixed avidin-biotin horseradish peroxidase complex solution (ABC Elite Kit, Vector Laboratories) for 60 minutes. The peroxidase complex was visualized by a 4 to 5-minute exposure to chromogen solution (0.05% 3,30-diaminobenzidine tetrahydrochloride (wt/vol, Sigma-Aldrich), 0.4 mg/ml nickel ammonium sulfate to produce a blue-black product. The reaction was stopped by washing in distilled water and PBS. Sections were dehydrated and cover-slipped with quick mounting medium (Eukitt, Fluka Analytical).

Zhang Z., Ferretti V., Güntan İ., Moro A., Steinberg E.A., Ye Z., Zecharia A.Y., Yu X., Vyssotski A.L., Brickley S.G., Yustos R., Pillidge Z.E., Harding E.C., Wisden W, & Franks N.P. (2015). Neuronal ensembles sufficient for recovery sleep and the sedative actions of α2 adrenergic agonists. Nature neuroscience, 18(4), 553-561.

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Immunohistochemical Analysis of Tissue Samples

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Sample sections were fixed in 10% buffered formalin, and were then embedded in paraffin and were mounted on slides. After deparaffinisation and rehydration, sample sections were soaked in 3% hydrogen peroxide to inactive endogenous peroxidase activity, and 10-mM sodium citrate (pH 6.0) was used for antigen retrieval by microwaving. Samples were then incubated 2.5% horse serum and with antibodies overnight at 4 ºC, and were detected using the Avidin-Biotin Complex System (Vector Laboratories).

Huang G., Hu H., Zhang Y., Zhu Y., Liu J., Tan B, & Chen T. (2019). Triptolide sensitizes cisplatin-resistant human epithelial ovarian cancer by inhibiting the phosphorylation of AKT. Journal of Cancer, 10(13), 3012-3020.

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Immunohistochemical Analysis of Colonic Tissue

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Colonic frozen sections (4 μm thick) were stained with anti-Ki67 (Abcam, Cambridge, MA, USA), rabbit anti-CHI3L1 (Affinity Bioreagent, Golden, CO), rat anti-F4/80 (Serotec, Raleigh, NC, USA), -CD4 (eBioscience, San Diego, CA, USA), -CD11b (BD Pharmingen, San Jose, CA, USA), or -cytokeratin 8, TROMA–1 (Developmental Studies Hybridoma Bank at the University of Iowa (Iowa City, IA) antibodies, as well as normal rabbit Ig using the avidin-biotin-complex system (Vector laboratories) as previously described [12 (link)].

Low D., DeGruttola A.K., Poltrak A., Mizoguchi A., Mino-Kenudson M, & Mizoguchi E. (2015). High Endogenous Expression of Chitinase 3-Like 1 and Excessive Epithelial Proliferation with Colonic Tumor Formation in MOLF/EiJ Mice. PLoS ONE, 10(10), e0139149.

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Neuronal Immunostaining of Brain Sections

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Coronal sections of the brain were cut at a thickness of 30 µm using a vibrating microtome (vibratome 1000, the Vibratome Company, St. Louis, MO, USA). Primary antibodies, including mouse anti-tyrosine hydroxylase (TH; 1:2000; Sigma-Aldrich) or mouse anti-Parvalbumin (PV) (1:2000; Sigma-Aldrich), were used. Biotinylated goat anti-mouse IgG (1:500 the Jackson ImmunoResearch Laboratories, West Grove, PA, USA) was used as the secondary antibody. Immunoreactivity was amplified using an avidin-biotin complex system (1:100, Vector Labs, Burlingame, CA, USA). TH is the key enzyme for dopamine synthesis, and TH immuno-histochemistry was used to label dopaminergic neurons. PV immunostaining was used to label PV-positive GABAergic interneurons. The densities of immunopositive cells were estimated using StereoInvestigator system (MicroBrightField Bioscience, Williston, VT, USA) [35 (link)].

Chang Y.C., Li W.Y., Lee L.J, & Lee L.J. (2020). Interplay of Prenatal and Postnatal Risk Factors in the Behavioral and Histological Features of a “Two-Hit” Non-Genetic Mouse Model of Schizophrenia. International Journal of Molecular Sciences, 21(22), 8518.

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Immunohistochemical Staining of Tumor Sections

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The resected tumours were fixed in 10% buffered formalin, embedded in paraffin and mounted on slides. After deparaffinization and rehydration, the tumour sections were incubated in 3% hydrogen peroxide to suppress endogenous peroxidase activity. Antigen retrieval was achieved by microwaving the sections in 10 mM sodium citrate solution (pH 6.0). Sections were then blocked by incubating with 2.5% horse serum, and then antibodies were applied overnight at 4ºC. Antibody detection was achieved using an avidin-biotin complex system (Vector Laboratories, Burlingame, CA). Slides were stained with 3,3′-diaminobenzidine, then washed and counterstained with hematoxylin. Slides were subsequently dehydrated, treated with xylene and mounted.

Zeng C., Chen T., Zhang Y, & Chen Q. (2017). Hedgehog signaling pathway regulates ovarian cancer invasion and migration via adhesion molecule CD24. Journal of Cancer, 8(5), 786-792.

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Visualizing Nuclear c-Fos Expression via DAB Staining

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