Non immune goat serum

Manufactured by Vector Laboratories

Non-immune goat serum is a liquid derived from the blood of goats. It is used as a reagent in various laboratory techniques, including immunohistochemistry, ELISA, and Western blotting, to block non-specific protein binding and reduce background staining.

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3 protocols using non immune goat serum

Immunohistochemical Analysis of Decalcified Femurs

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Formalin-fixed femurs were processed as previously described (14 (link)). Briefly, bones were decalcified, embedded in paraffin, and sectioned on a rotary microtome at 4 μm. Sections were placed on adhesive slides, deparaffinized, and submitted to antigen-retrieval protocols (Supplementary Table 2). Following pretreatments, standard avidin-biotin complex staining steps were performed at room temperature using a Dako Autostainer. After blocking nonspecific protein with nonimmune goat serum (Vector Laboratories, Burlingame, CA), sections were incubated with an avidin (Vector Laboratories)/biotin (Sigma-Aldrich) blocking system. Sections were incubated with antibodies (Supplementary Table 3) followed by R.T.U. VectaStain Elite ABC Reagent (Vector) and were developed with NovaRED (Vector Laboratories) followed by counterstain in Gill 2 hematoxylin (Richard Allen, Kalamazoo, MI). Slides then were dehydrated, cleared, and placed on a slide under a coverslip with Flotex Permount mounting media.

Dominguez JM I.I., Yorek M.A, & Grant M.B. (2014). Combination Therapies Prevent the Neuropathic, Proinflammatory Characteristics of Bone Marrow in Streptozotocin-Induced Diabetic Rats. Diabetes, 64(2), 643-653.

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Immunofluorescence Staining of PTGFR

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After antigen retrieval with 10 mM sodium citrate (pH 6), slides were blocked with 5% non-immune goat serum (Vector Laboratories, Burlingame, CA) in PBS containing 0.1% Triton; all antibody solutions were made in this blocking buffer. Slides were incubated for 1 hour at room temperature either with primary antibody generated against PTGFR (5 μg/ml; Cayman) or no primary antibody, followed by incubation with Alexa Fluor 488-conjugated anti-rabbit secondary antibody (4 μg/ml; Molecular Probes, Eugene, OR). The slides were treated with 1% Sudan Black in 70% methanol to reduce autofluorescence and coverslipped in Vectashield medium containing propidium iodide (Vector). In some experiments, the anti-PTGFR primary antibody was pre-incubated with the blocking peptide at a 1:2 ratio prior to use in the primary antibody incubation step.
Conventional fluorescence images were obtained using an Olympus BX41 fluorescent microscope fitted with a DP70 digital camera and associated software (Olympus, Melville, NY). Confocal laser microscopy was performed using a Zeiss 510 laser scanning confocal microscope with LSM5 software for image acquisition (Carl Zeiss Inc., Thornwood, NY) using 488 nm excitation with a 505/550 band pass filter (green channel) and 543 nm excitation with a 560 nm long pass filter (red channel).

Kim S.O., Markosyan N., Pepe G.J, & Duffy D.M. (2015). Estrogen Promotes Luteolysis by Redistributing Prostaglandin F2α Receptors Within Primate Luteal Cells. Reproduction (Cambridge, England), 149(5), 453-464.

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ALP and Nitrotyrosine Immunohistochemistry

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Tissue sections were stained with hematoxylin and eosin. For ALP activity staining, the sections were stained with 0.1% naphthol AS-MX phosphate (Sigma-Aldrich) and 0.1% fast blue BB salt (Sigma-Aldrich) in 0.1 M Tris-buffer (pH 9.2) at 37 °C for 30 min^{14 (link)}. Immunohistochemistry with anti-nitrotyrosine antibody was performed as described previously^{14 (link)}. The sections were quenched with 0.3% H₂O₂ in PBS for 60 min and blocked with 10% non-immune goat serum (Vector Laboratories) for 30 min. Then, the samples were incubated with anti-nitrotyrosine antibody (1:100; Millipore) at 4 °C overnight. Next, the sections were incubated with a biotinylated antibody (1:200; Vector Laboratories) for 45 min, followed by avidin-biotin complex (1:100; Vector Laboratories) for 60 min. The sections were reacted with 0.02% 3,3″-diaminobenzidine tetrahydrochloride (Dojindo Laboratories) and 0.06% H₂O₂ in 0.05 M Tris buffer, pH 7.6 and counter-stained with hematoxylin. Immunohistochemical controls were incubated with non-immune rabbit IgG (Vector Laboratories) instead of the primary antibody. The sections were observed under an Axio Imager M2 microscope.

Sonoda S., Mei Y.F., Atsuta I., Danjo A., Yamaza H., Hama S., Nishida K., Tang R., Kyumoto-Nakamura Y., Uehara N., Kukita T., Nishimura F, & Yamaza T. (2018). Exogenous nitric oxide stimulates the odontogenic differentiation of rat dental pulp stem cells. Scientific Reports, 8, 3419.

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