Biotin block

Manufactured by Vector Laboratories

Sourced in United States

Biotin Block is a product designed to reduce non-specific binding of biotin-based detection systems in immunohistochemistry and other applications. It is a ready-to-use solution that can be applied to tissue sections or cell preparations to effectively block endogenous biotin.

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Lab products found in correlation

Avidin block, by Vector Laboratories (6 mentions) Peroxidase block, by Agilent Technologies (3 mentions) Streptavidin hrp, by Biocare Medical (2 mentions) Goat anti rabbit biotinylated igg, by Biocare Medical (2 mentions) Diaminobenzadine, by Agilent Technologies (2 mentions) Protein block serum free, by Agilent Technologies (2 mentions) Normal goat serum (ngs), by Thermo Fisher Scientific (1 mentions) Eukitt mounting medium, by Merck Group (1 mentions) Vectastain elite abc kit, by Vector Laboratories (1 mentions) Streptavidin horseradish peroxidase, by Biocare Medical (1 mentions) Diaminobenzidine, by Agilent Technologies (1 mentions) Mayer s hematoxylin, by Agilent Technologies (1 mentions) Aec substrate, by Abcam (1 mentions) Q color5 imaging system, by Olympus (1 mentions)

6 protocols using biotin block

Kidney Damage and Glut2 Staining Protocol

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Formalin-fixed kidney sections were cut at 4 μm slices, dried and deparaffinized for subsequent streptavidin-biotin immunohistochemistry. After deparaffinization, the slides were treated with a citrate buffer (pH 6) for a total of 35 min. The slides were blocked with a peroxidase block (DAKO, Carpinteria, CA, USA), Avidin Block (Vector Labs, Burlingame, CA, USA), Biotin Block (Vector Labs), and serum-free Protein Block (DAKO). For kidney damage analysis, the tissue was stained with Masson's trichrome. For Glut2 staining, liver sections were embedded in one paraffin block for tissue microarrays and slices were incubated 1:200 overnight with anti-Glut2 antibodies (#sc-9117, Santa Crus Biotech, Dallas, TX). Secondary detection was performed with Goat anti-Rabbit biotinylated IgG (Biocare, Concord, CA, USA) followed by Streptavidin HRP (Biocare) and visualized with Diaminobenzadine (DAB, DAKO). ImageJ software was used to assess intensity of Glut2 staining in hepatocytes. Average signal levels in 10 areas in each liver were measured, and background (counterstained samples treated with secondary antibodies) was subtracted.

Pavlov T.S., Levchenko V., Ilatovskaya D.V., Li H., Palygin O., Pastor-Soler N.M., Hallows K.R, & Staruschenko A. (2017). Lack of Effects of Metformin and AICAR Chronic Infusion on the Development of Hypertension in Dahl Salt-Sensitive Rats. Frontiers in Physiology, 8, 227.

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Immunohistochemical Analysis of Kidney Tissue

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Extracted kidneys were placed into a neutral buffered 10% formalin solution. The kidney sections were cut at 4 μm slices, dried, and deparaffinized for subsequent streptavidin-biotin immunohistochemistry. After deparaffinization, the slides were treated with a citrate buffer pH6 for a total of 35 min. The slides were blocked with a peroxidase block (DAKO, Carpinteria, California, USA), Avidin Block (Vector Labs, Burlingame, California, USA), Biotin Block (Vector Labs), and serum-free Protein Block (DAKO). For morphological analysis, the tissue was stained with hematoxylin-eosin. For immunohistochemical staining, tissue sections were incubated for 30 min with anti-α, β or γ-ENaC antibodies. Secondary detection was performed with goat anti-rabbit biotinylated immunoglobulin G (IgG) (Biocare, Concord, California, USA) followed by streptavidin conjugated with horseradish peroxidase (Biocare) and visualized with diaminobenzadine (DAKO).

Pavlov T.S., Levchenko V., Ilatovskaya D.V., Moreno C, & Staruschenko A. (2016). Renal sodium transport in renin-deficient Dahl salt-sensitive rats. Journal of the Renin-Angiotensin-Aldosterone System: JRAAS, 17(3), 1470320316653858.

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Immunohistochemical Analysis of Parvalbumin Neurons

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Fixation of the brain was performed by transcardially perfusing 0.1M phosphate-buffered saline (PBS) followed by 4% paraformaldehyde on the 10^th day after drug or saline exposure. Brains were cryoprotected, flash frozen and hippocampi sectioned into 50 μm thick slices. Sections were treated with 1% H₂O₂ for 30 min at room temperature (RT). The sections were blocked for 2 hr at RT with 10% normal goat serum (Life Technologies) in 0.1M PBS containing 0.5% Triton-X 100 and Avidin block (4 drops/mL; Vector Laboratories). For PV immunostaining, 50μm thick sections were incubated for 36 hr at 4°C with a mouse monoclonal PV antibody (1:2000; Swant, cat# PV235) in 0.1 M PBS containing 0.5% Triton-X 100, 1% NGS and Biotin block (4 drops/ml; Vector Laboratories). Sections were then incubated in goat anti-mouse biotinylated secondary antibodies (Millipore, cat# AP124b) for 2hr at RT followed by incubation in avidin-biotin- horseradish peroxidase (Vectastain Elite ABC Kit; Vector Laboratories) for 2hr at RT. Sections were then developed in diaminobenzidine (DAB/Metal Concentrate; Pierce) and mounted on glass slides and coverslipped with Eukitt mounting medium (Sigma- Aldrich). All slides were coded and the code was not broken until the end of quantitative analysis.

Collins S.A., Gudelsky G.A, & Yamamoto B.K. (2015). MDMA-induced loss of parvalbumin interneurons within the dentate gyrus is mediated by 5HT2A and NMDA receptors. European journal of pharmacology, 761, 95-100.

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Immunohistochemical Analysis of Rat Kidney

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Rat kidneys were fixed in 10% formalin and processed for paraffin embedding as previously described^{62 (link)}. Kidney sections were cut at 4 μm, dried, and deparaffinized for subsequent labeling by streptavidin-biotin immunohistochemistry. After deparaffinization, slides were treated with a citrate buffer (pH 6) for total of 35 min. Slides were blocked with a perioxidase block (Dako, Coppenhagen, Denmark), avidin block (Vector Laboratories, Burlingame, CA), biotin block (Vector Laboratories), and serum-free protein block (Dako). Tissue sections were incubated for 90 min in antibody to Kidney Injury Molecule-1 (Rat KIM-1Ab, 1:300, #AF3689, R&D Systems, Inc) or megalin (lipoprotein-related protein 2 (LRP2) Ab, 1:2500, from Dr. Franziska Theilig University of Kiel, Kiel, Germany). Secondary detection was performed with goat anti-goat or anti-rabbit biotinylated IgG (Biocare, Tempe, AZ) followed by streptavidin-horseradish peroxidase (Biocare) and visualized with diaminobenzidine (Dako). All slides were counterstained with Mayer’s hematoxylin (Dako), dehydrated, and mounted with a permanent mounting medium (Sakura, Torrance, CA).

Rinschen M.M., Palygin O., El-Meanawy A., Domingo-Almenara X., Palermo A., Dissanayake L.V., Golosova D., Schafroth M.A., Guijas C., Demir F., Jaegers J., Gliozzi M.L., Xue J., Hoehne M., Benzing T., Kok B.P., Saez E., Bleich M., Himmerkus N., Weisz O.A., Cravatt B.F., Krüger M., Benton H.P., Siuzdak G, & Staruschenko A. (2022). Accelerated lysine metabolism conveys kidney protection in salt-sensitive hypertension. Nature Communications, 13, 4099.

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Immunohistochemical Analysis of Renal ENaC

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Extracted kidneys were placed into a neutral buffered 10% Formalin solution. The kidney sections were cut at 4 μm slices, dried and deparaffinized for subsequent streptavidin-biotin immunohistochemistry. After deparaffinization, the slides were treated with a citrate buffer pH6 for total of 35 min. The slides were blocked with a peroxidase block (DAKO, Carpinteria, CA, USA), Avidin Block (Vector Labs, Burlingame, CA, USA), Biotin Block (Vector Labs), and serum-free Protein Block (DAKO). For morphological analysis, the tissue was stained with haematoxylin-eosin. For immunohistochemical staining, tissue sections were incubated for 30 min with anti-α, β or γ-ENaC antibodies. Secondary detection was performed with Goat anti-Rabbit biotinylated IgG (Biocare, Concord, CA, USA) followed by Streptavidin HRP (Biocare) and visualized with Diaminobenzadine (DAKO).

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Quantifying Macrophages in Aortic Tissue

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5 μM sections of the proximal aorta were fixed in cold acetone and incubated at room temperature in 2% BSA/PBS. Slides were treated with avidin block (Vector, Olean, NY), biotin block (Vector), and peroxidase-activity block (9:1 ratio of methanol:30% H₂O₂). Macrophages were stained using a 1:25 dilution of rat-anti mouse macrophage/monocyte, clone MOMA-2 (MilliporeSigma) in 2% BSA/PBS for 1 hour at RT. Next, slides were stained with a 1:200 dilution of biotin goat-anti rat (BD Biosciences) in 2% BSA/PBS for 30 minutes at 37°C.
Strepavadin-HRP (Biogenex, Fremont, CA) was applied for 20 minutes followed by AEC substrate (Abcam) for 2 minutes. Hematoxylin counterstain was applied for 2 minutes and slides were imaged using a Q-Color5™ imaging system (Olympus, Center Valley, PA). Quantification of macrophage/monocyte area was performed using ImageJ and averaged for four sections.

Marvin J., Rhoads J.P, & Major A.S. (2019). FcγRIIb on CD11c+ cells modulates serum cholesterol and triglyceride levels and differentially affects atherosclerosis in male and female Ldlr−/− mice. Atherosclerosis, 285, 108-119.

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