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Biotinylated goat anti rat

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Biotinylated goat anti-rat is a secondary antibody reagent produced by Vector Laboratories. It is designed to detect and bind to rat primary antibodies, with biotin conjugated to the goat anti-rat IgG for downstream detection and applications.

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Lab products found in correlation

Biotinylated goat anti rabbit, by Vector Laboratories (3 mentions) Avidin biotin blocking kit, by Vector Laboratories (2 mentions) Antigen unmasking solution, by Vector Laboratories (2 mentions) Vectastain abc kit, by Vector Laboratories (2 mentions) Rabbit anti human nrp1 44 2, by Abcam (2 mentions) Rabbit monoclonal anti mouse nrp1 clone epr3113, by Abcam (2 mentions) Monoclonal anti pcna clone pc10, by Agilent Technologies (2 mentions) Monoclonal rat anti mouse cd31 clone mec13.3, by BD (2 mentions) Hrp conjugated rat anti mouse igg2a, by Bio-Rad (2 mentions) Vectastain elite hrp linked avidin, by Vector Laboratories (2 mentions) Rabbit anti human keratin 1 af87, by Fortrea (2 mentions) Rabbit anti human keratin 14 af64, by Fortrea (2 mentions) Immpact dab hrp substrate, by Vector Laboratories (1 mentions) Goat anti mouse igg hrp, by Santa Cruz Biotechnology (1 mentions) F4 80, by Bio-Rad (1 mentions) Goat anti rabbit igg hrp, by Abcam (1 mentions) Olive oil, by Merck Group (1 mentions) Carbon tetrachloride, by Merck Group (1 mentions) Ketaved, by Vedco (1 mentions) Buprenex, by Reckitt Benckiser (1 mentions)

10 protocols using biotinylated goat anti rat

1

Histopathological and Immunohistochemical Analysis of Tumor Microenvironment

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HT was confirmed by histopathology. HT exposed tumor tissues (n=3) were fixed in 10% neutral buffered formalin, processed, and embedded in paraffin 48 (link). Histopathological examinations of 4 μm sections stained with hematoxylin and eosin (H&E) were performed by a veterinary pathologist. To examine the tumor immune environments, the tumor sections from mice bearing bilateral tumors were stained for CD3 antibody for immunohistochemical analysis. Briefly, 10% neutral buffered formalin fixed treated and untreated (abscopal) tumors were embedded in paraffin and cut into 4 μm sections. Antigen retrieval was performed in a decloaking chamber. Normal goat serum was used for blocking. Incubation with the anti-rat CD3 primary antibody (Abcam, Cambridge, MA) was performed at 4°C overnight. Biotinylated goat anti-rat (Vector Laboratories, Burlingame, CA) was used as the secondary antibody and detected with ImmPACT DAB HRP Substrate (Vector Laboratories, Burlingame, CA).
Singh M.P., Sethuraman S.N., Miller C., Malayer J, & Ranjan A. (2021). Boiling histotripsy and in-situ CD40 stimulation improve the checkpoint blockade therapy of poorly immunogenic tumors. Theranostics, 11(2), 540-554.
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2

Molecular Markers in Liver Injury

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Olive oil and carbon tetrachloride were purchased from Sigma-Aldrich (St. Louis, MO) and Buprenex (buprenorphine HCL) was manufactured by Reckitt Benckiser Healthcare (UK, Ltd, Hull England) and distributed by Reckitt Benckiser Pharmaceuticals, Inc (Richmond, VA). The anesthetic used was a mixture containing Ketamine (Akom, Inc, Decator, IL), Xylazine (KetaVed, VedCO, Inc., St. Joseph, MO), and Acepromazine (VedCO, Inc).
Primary antibodies used include: Cytochrome P450 2E1 (CYP2E1, ab28146, AbCam, Cambridge, MA), Proliferating cell nuclear antigen (PCNA, clone PC10, EMD-Millipore, Billerica, MA), Cyclin D1 (CCND1, clone 92g2, Cell Signaling, Danvers, MA), α-smooth muscle actin (αSMA, clone 1A4, AbCam), Ki67 (ab66155, AbCam), Glyceraldehyde 3-phosphate dehydrogenase (GAPDH, clone 14C10, Cell Signaling), F4/80 (Bio-Rad, MCA497), NQO1 (Sigma-Aldrich, N5288), goat anti-mouse IgG-HRP (sc-2005, Santa Cruz, Santa Cruz, CA), goat anti-rabbit IgG-HRP (ab97080, AbCam), biotinylated goat anti-rat (Vector), and donkey anti-rabbit IgG-Alexa Flour 488 conjugate (a-21206, Life Technologies, Waltham, MA).
McCracken J.M., Chalise P., Briley S.M., Dennis K.L., Jiang L., Duncan F.E, & Pritchard M.T. (2017). C57BL/6 Substrains Exhibit Different Responses to Acute Carbon Tetrachloride Exposure: Implications for Work Involving Transgenic Mice. Gene Expression, 17(3), 187-205.
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3

CD44 and PAS Immunostaining Protocol

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Immunohistochemistry was performed on 4-μm paraffin-embedded or 3% paraformaldehyde-fixed cryosections. CD44 immunostaining were combined with a PAS staining. Sections were blocked with Avidin/Biotin Blocking Kit (Vector Laboratories) and 3% H2O2. The sections were subjected to microwave antigen retrieval in Antigen Unmasking Solution (Vector Laboratories) followed by incubation with the primary (anti-CD44) and secondary antibodies. As secondary antibodies, we used biotinylated goat anti-rat (Vector Laboratories). Detection was carried out with Vectastain ABC kit (Vector Laboratories) with the use of peroxidase as label and 3,3-diaminobenzidine as substrate and nickel chloride enhancement. Subsequent to the immunostaining, a PAS staining was performed.
Kuppe C., Leuchtle K., Wagner A., Kabgani N., Saritas T., Puelles V.G., Smeets B., Hakroush S., van der Vlag J., Boor P., Schiffer M., Gröne H.J., Fogo A., Floege J, & Moeller M.J. (2019). Novel parietal epithelial cell subpopulations contribute to focal segmental glomerulosclerosis and glomerular tip lesions. Kidney international, 96(1), 80-93.
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4

Immunohistochemical Analysis of Tissue Sections

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Formalin-fixed-paraffin-embedded tissue sections were cut at a thickness of 5 µm and rehydrated. Heat-induced epitope retrieval was performed in sodium citrate antigen retrieval buffer. Tissues were stained with H&E or, for immunohistochemistry, Granzyme B (Abcam, ab4059; Cambridge, MA), Ki-67 (Abcam, Ab15580), PD1 (Cell Signaling 9664S), cleaved caspase 3 (Cell Signaling, 84651S), PD-L1 (Invitrogen, 14-5982-82; Waltham, MA), CD4 (Invitrogen, 14-0042-82), CD8 (Invitrogen, 14-0808-82) and CXCL9 (BioXCell, BE0309). Following the addition of primary antibody, the tissues were incubated with biotinylated goat anti-rabbit (Vector Laboratories, BA-1000; Burlingame, CA), biotinylated goat anti-hamster (Invitrogen, 31750), biotinylated goat anti-rat (Vector Laboratories, BA-9401) IgG (H + L) secondary antibodies or, for immunofluorescent staining, Alexa Fluor 488-conjugated goat anti-rabbit (Invitrogen, A11034) and Alexa Fluor 555 goat anti-rat (Invitrogen, A21434) with a DAPI counterstain. Regions of positive staining were quantified using ImageJ [26 (link)].
Anderson K., Ryan N., Nedungadi D., Lamenza F., Swingler M., Siddiqui A., Satoskar A., Upadhaya P., Pietrzak M, & Oghumu S. (2022). STAT1 is regulated by TRIM24 and promotes immunosuppression in head and neck squamous carcinoma cells, but enhances T cell antitumour immunity in the tumour microenvironment. British Journal of Cancer, 127(4), 624-636.
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5

CD44 and PAS Immunostaining Protocol

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Immunohistochemistry was performed on 4-μm paraffin-embedded or 3% paraformaldehyde-fixed cryosections. CD44 immunostaining were combined with a PAS staining. Sections were blocked with Avidin/Biotin Blocking Kit (Vector Laboratories) and 3% H2O2. The sections were subjected to microwave antigen retrieval in Antigen Unmasking Solution (Vector Laboratories) followed by incubation with the primary (anti-CD44) and secondary antibodies. As secondary antibodies, we used biotinylated goat anti-rat (Vector Laboratories). Detection was carried out with Vectastain ABC kit (Vector Laboratories) with the use of peroxidase as label and 3,3-diaminobenzidine as substrate and nickel chloride enhancement. Subsequent to the immunostaining, a PAS staining was performed.
Kuppe C., Leuchtle K., Wagner A., Kabgani N., Saritas T., Puelles V.G., Smeets B., Hakroush S., van der Vlag J., Boor P., Schiffer M., Gröne H.J., Fogo A., Floege J, & Moeller M.J. (2019). Novel parietal epithelial cell subpopulations contribute to focal segmental glomerulosclerosis and glomerular tip lesions. Kidney international, 96(1), 80-93.
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6

Immunohistochemistry of Nrp1 and Markers

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Formalin-fixed, paraffin-embedded sections were deparaffinized in xylene and rehydrated through a graded series of alcohols to water. Antigen retrieval included proteinase K (20 μg/ml) for CD31 and mouse Nrp1 and heat-induced epitope retrieval for PCNA. Staining with the other antibodies did not require antigen retrieval.
Endogenous peroxidase was inhibited with 3% H2O2 in methanol, and proteins were blocked in TNB. Sections were incubated in primary antibodies overnight at 4°C including rabbit anti-human NRP1 44-2 (not commercial), rabbit monoclonal anti-mouse Nrp1 (clone EPR3113; detects C terminus and not sNrp1; Epitomics), monoclonal anti-PCNA (clone PC10; Dako), rabbit anti-human keratin 1 (AF87; Covance), rabbit anti-human keratin 14 (AF64; Covance), and monoclonal rat anti-mouse CD31 (clone MEC13.3; BD Pharmingen). Sections were incubated in secondary antibodies including HRP-conjugated rat anti-mouse IgG2a (Serotec), biotinylated goat anti-rabbit (Vector), biotinylated goat anti-rat (mouse absorbed; Vector), Vectastain Elite HRP-linked avidin, and DAB. Cells were counterstained with hematoxylin and mounted with Permount.
Shahrabi-Farahani S., Wang L., Zwaans B.M., Santana J.M., Shimizu A., Takashima S., Kreuter M., Coultas L., D'Amore P.A., Arbeit J.M., Akslen L.A, & Bielenberg D.R. (2014). Neuropilin 1 expression correlates with differentiation status of epidermal cells and cutaneous squamous cell carcinomas. Laboratory investigation; a journal of technical methods and pathology, 94(7), 752-765.
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7

Immunohistochemistry of Nrp1 and Markers

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Formalin-fixed, paraffin-embedded sections were deparaffinized in xylene and rehydrated through a graded series of alcohols to water. Antigen retrieval included proteinase K (20 μg/ml) for CD31 and mouse Nrp1 and heat-induced epitope retrieval for PCNA. Staining with the other antibodies did not require antigen retrieval.
Endogenous peroxidase was inhibited with 3% H2O2 in methanol, and proteins were blocked in TNB. Sections were incubated in primary antibodies overnight at 4°C including rabbit anti-human NRP1 44-2 (not commercial), rabbit monoclonal anti-mouse Nrp1 (clone EPR3113; detects C terminus and not sNrp1; Epitomics), monoclonal anti-PCNA (clone PC10; Dako), rabbit anti-human keratin 1 (AF87; Covance), rabbit anti-human keratin 14 (AF64; Covance), and monoclonal rat anti-mouse CD31 (clone MEC13.3; BD Pharmingen). Sections were incubated in secondary antibodies including HRP-conjugated rat anti-mouse IgG2a (Serotec), biotinylated goat anti-rabbit (Vector), biotinylated goat anti-rat (mouse absorbed; Vector), Vectastain Elite HRP-linked avidin, and DAB. Cells were counterstained with hematoxylin and mounted with Permount.
Shahrabi-Farahani S., Wang L., Zwaans B.M., Santana J.M., Shimizu A., Takashima S., Kreuter M., Coultas L., D'Amore P.A., Arbeit J.M., Akslen L.A, & Bielenberg D.R. (2014). Neuropilin 1 expression correlates with differentiation status of epidermal cells and cutaneous squamous cell carcinomas. Laboratory investigation; a journal of technical methods and pathology, 94(7), 752-765.
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8

Immunohistochemical Staining of Brain Tissue

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Sections of brain tissue were washed in 0.15 ​M ​PB and blocked with 3% normal goat serum (GIBCO, Carlsbad, CA). For BrdU, antigen retrieval was performed by incubating free floating tissue sections in 4 ​N HCl for 20 ​min ​at room temperature. Staining of free-floating sections was performed using rabbit anti-P2Y12R (1:3000; Anaspec, Fremont, CA), rat anti-BrdU (1:300; Abcam, Cambridge, UK), and rat anti-CD68 (1:1000; Serotec, Raleigh, NC). Secondary antibodies included goat anti-rabbit Alexa 594 (1:2000; Invitrogen) and goat anti-rat Alexa 488 (1:2000; Invitrogen). To visualize BrdU for Fig. 1B, biotinylated goat anti-rat (1:1000; Vector, Burlingame, CA), followed by avidin-biotin complex kit solution (Elite Vectastain ABC, Vector) and subsequent 3-3’ -diaminobenzidine reaction kits (Vector), were used according to the manufacturer’s protocols. Hemoxylin and eoxin (H and E) were used to stain the tumor sections presented in Fig. 7B.
Belcher E.K., Sweet T.B., Karaahmet B., Dionisio-Santos D.A., Owlett L.D., Leffler K.A., Janelsins M.C., Williams J.P., Olschowka J.A, & O’Banion M.K. (2020). Cranial irradiation acutely and persistently impairs injury-induced microglial proliferation. Brain, Behavior, & Immunity - Health, 4, 100057.
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9

BrdU Immunohistochemistry for Dividing Cells

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After overnight fixation in 4% paraformaldehyde, the tissue was transferred into 30% sucrose solution. Brains were sectioned at 40 μm using a cryostat. A one-in-six series was stained for bromodeoxyuridine (BrdU) to identify newly divided cells. Immunohistochemistry was performed according to Kohman et al. [59 ] using rat anti-BrdU (1:100; AbD Serotec, Raleigh, NC) as the primary antibody and biotinylated goat anti-rat (1:250; Vector Laboratories, Burlingame, CA) as the secondary antibody. After incubation in the secondary antibody, sections were then treated with the avidin/biotinylated enzyme complex ABC system (Vector, Burlingame, CA) and stained using a diaminobenzidine kit (DAB; Sigma, St. Louis, MO).
Gibbons T.E., Pence B.D., Petr G., Ossyra J.M., Mach H.C., Battacharya T.K., Perez S., Martin S.A., McCusker R.H., Kelley K.W., Rhodes J.S., Johnson R.W, & Woods J.A. (2014). Voluntary wheel running, but not a diet containing (-)-epigallocatechin-3-gallate and β-alanine, improves learning, memory and hippocampal neurogenesis in aged mice. Behavioural brain research, 272, 131-140.
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10

Quantifying CD4+ T Cells in Tissue

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Formalin-fixed paraffin embedded sections underwent heat induced epitope retrieval. Rat anti mouse CD4 (4SM95, ThermoFisher) was used as a primary antibody followed by biotinylated goat anti-rat (Vector, Burlingame, CA) and ABC-HRP. Staining was developed using DAB/peroxide solution (ThermoFisher). Slides were counterstained with 50% Gill’s hematoxylin diluted in with water and then cleared. For quantification, 4 sections from each graft were obtained, at 100 μm intervals. FIJI color deconvolution and thresholding techniques were used to isolate DAB signal, and FIJI particle analysis was used to quantitate CD4 cells.
Williams L., Bagley J, & Iacomini J. (2021). The role of IL-6 in hyperlipidemia induced accelerated rejection. American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons, 22(2), 427-437.
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