Goat or donkey serum

Manufactured by Jackson ImmunoResearch

Sourced in United States

Goat or donkey serum is a biological product used as a general diluent or blocking agent in various immunoassays and immunohistochemical techniques. It is derived from the blood of either goats or donkeys.

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Lab products found in correlation

4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (4 mentions) Eclipse 700, by Nikon (3 mentions) Volocity image analysis software, by PerkinElmer (3 mentions) Rat anti f4 80 monoclonal antibody, by Abcam (2 mentions) Goat polyclonal anti mcp 1 antibody, by Santa Cruz Biotechnology (2 mentions) Coverslip, by Thermo Fisher Scientific (2 mentions) Fluorescence labeled secondary antibody, by Jackson ImmunoResearch (1 mentions) Ctr6500, by Leica Microsystems (1 mentions) Fluorescent dye, by Thermo Fisher Scientific (1 mentions) Dylight 488 conjugated lycopersicon esculentum lectin, by Vector Laboratories (1 mentions) Lsm 710, by Zeiss (1 mentions) Casein, by Vector Laboratories (1 mentions) Prolong gold antifade, by Thermo Fisher Scientific (1 mentions) Triton x 100, by Merck Group (1 mentions) Bovine serum albumin (bsa), by Gemini Bio (1 mentions) Foxp3, by Thermo Fisher Scientific (1 mentions) Cd11c, by BD (1 mentions) Bovine serum albumin (bsa), by Cell Signaling Technology (1 mentions) A confocal microscope, by Olympus (1 mentions) 8 well glass chamber slides, by Thermo Fisher Scientific (1 mentions)

10 protocols using goat or donkey serum

Immunohistochemical Analysis of Inflammation in Intracranial Aneurysms

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Immunohistochemical analyses were performed as previously described [19 (link)]. Briefly, at the indicated period after aneurysm induction, 5-um-thick frozen sections were made as described above. After blocking with 3% donkey or goat serum (Jackson ImmunoResearch, Baltimore, MD, USA), slices were incubated with primary antibodies followed by incubation with fluorescence-labeled secondary antibodies (Jackson ImmunoResearch). Finally, fluorescent images were acquired through a confocal fluorescence microscope system (CTR6500, Leica Microsystems, Tokyo, Japan). Representative images from at least 3 independent samples are shown in Figures 1 and 2. The relative intensity of positive staining in IA walls from each experiment was measured by imaging software and statistically analyzed.
The following primary antibodies were used: mouse monoclonal anti-smooth muscle alpha actin antibody (Thermo scientific, Waltham, MA, USA), rabbit monoclonal anti-phospho NF-kappaB p65 (ser536) antibody (Cell Signaling Technology, Danvers, MA, USA), rat monoclonal anti-TNF-alpha antibody (Lifespan Bioscience, Seattle, WA, USA), goat polyclonal anti-MCP-1 antibody (Santa Cruz Biotechnology, Dallas, TX, USA), rabbit polyclonal anti-COX-2 antibody (Cayman Chemicals, Ann Arbor, MI, USA), rat monoclonal anti-F4/80 antibody (Abcam, Cambridge, UK).

Aoki T., Fukuda M., Nishimura M., Nozaki K, & Narumiya S. (2014). Critical role of TNF-alpha-TNFR1 signaling in intracranial aneurysm formation. Acta Neuropathologica Communications, 2, 34.

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Immunofluorescence Staining of IA Tissue

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IA specimen was harvested as described above and slices of 5 μm thickness were prepared. After being blocked with 3% donkey or goat serum (Jackson ImmunoResearch, Baltimore, MD), the slices were incubated with primary antibodies followed by incubation with secondary antibodies conjugated with fluorescent dye (Invitrogen, Waltham, MA). In some experiments, DyLight488‐conjugated

Lycopersicon esculentum

Lectin (Vector Laboratories, Burlingame, CA) was used instead of an antibody to visualize endothelial cells. Finally, immunofluorescence images were acquired on a confocal fluorescence microscope system (LSM‐710, Carl Zeiss Microscopy GmBH, Gottingen, Germany).
Primary antibodies used were; mouse monoclonal anti‐smooth muscle α actin antibody (#MS113, Thermo Fisher Scientific, Waltham, MA), rat monoclonal anti‐F4/80 antibody (#ab16911, Abcam, Cambridge, UK), goat polyclonal anti‐MCP‐1 antibody (#sc‐1785, Santa Cruz Biotechnology, Dallas, TX), rabbit polyclonal anti‐S1P1 antibody (#sc‐25489, Santa Cruz Biotechnology).

Yamamoto R., Aoki T., Koseki H., Fukuda M., Hirose J., Tsuji K., Takizawa K., Nakamura S., Miyata H., Hamakawa N., Kasuya H., Nozaki K., Hirayama Y., Aramori I, & Narumiya S. (2017). A sphingosine‐1‐phosphate receptor type 1 agonist, ASP4058, suppresses intracranial aneurysm through promoting endothelial integrity and blocking macrophage transmigration. British Journal of Pharmacology, 174(13), 2085-2101.

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Multiplex RNA and Immunofluorescence Staining

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Immunofluorescence staining was performed after the last RNA target was developed (step Amp4 in the RNAscope Multiplex Fluorescent Assay version 1, TSA amplification step in the RNAscope Multiplex Fluorescent Assay version 2), the tissue sections were washed with PBS and blocked with 5% donkey or goat serum (Jackson ImmunoResearch), 2% BSA (Gemini Bio-Products) + 0.2% Triton X-100 (SigmaAldrich) in 5X Casein (Vector Laboratories) for 30 min, followed by incubation with the mouse anti-huCD19 antibody to stain tumor cells (dilution 1:100,Clone ID: UMAB103, Origene) overnight at 4 °C.
Slides were washed, incubated with secondary donkey anti-mouse IgG-Alexa 647 or Alexa 488 (1:100; Molecular Probes/ThermoFisher Scientific) or a goat anti-mouse BV480 (1:100; Becton Dickinson) for 1 h at room temperature, and washed 2 times for 5 min in PBS + tween (0.05% v/v). Subsequently, samples were counterstained with WGA and DAPI (ACDbio) and mounted with ProLong Gold antifade (Thermofisher).

Eichholz K., Li A.Z., Diem K., Jensen M.C., Zhu J, & Corey L. (2021). A CAR RNA FISH assay to study functional and spatial heterogeneity of chimeric antigen receptor T cells in tissue. Scientific Reports, 11, 12921.

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Immunohistochemical Analysis of Lymphoid Tissues

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The following primary antibodies were used for immunohistochemistry diluted 1:200: ERTR7 (scbt, clone sc-73355), CD11c (BD, clone HL3), Laminin alpha 4 (Novus Bio, clone 775830), laminin alpha 5 (Novus Bio, polyclonal), Foxp3 (ThermoFisher, clone NRRF-30), Y-Ae (scbt, clone sc-32247). For immunohistochemistry, LNs and spleens were frozen with OCT compound (Scigen Tissue-Plus). Frozen sections were cut at 6μm, fixed with cold acetone, blocked with 5% goat or donkey serum (Jackson ImmunoResearch, West Grove, PA) and incubated with the indicated antibodies and DAPI. Samples were incubated with secondary antibodies diluted 1:400 for 1 h (Rabbit IgG, Goat polyclonal, Jackson Immunoresearch; Rat IgG Goat Polyclonal, Jackson Immunoresearch; Rabbit IgG, Donkey Polyclonal, Jackson Immunoresearch; Rat IgM, Goal Polyclonal Jackson Immunoresearch). Samples were further processed as described previously^{45 (link),46 (link),56 (link)}. Images were acquired with a Nikon Eclipse 700 (Nikon, Melville, NY, USA) and analyzed with Volocity image analysis software (version 4.7.2) Perkin Elmer, Waltham, MA). The positive staining area percentage was quantified based on at least three independent experiments with 3 sections per LN and 3–5 fields/section. Number of mice are indicated in figure legends.

Gammon J.M., Carey S.T., Saxena V., Eppler H.B., Tsai S.J., Paluskievicz C., Xiong Y., Li L., Ackun-Farmmer M., Tostanoski L.H., Gosselin E.A., Yanes A.A., Zeng X., Oakes R.S., Bromberg J.S, & Jewell C.M. (2023). Engineering the lymph node environment promotes antigen-specific efficacy in type 1 diabetes and islet transplantation. Nature Communications, 14, 681.

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Immunohistochemistry and Whole Mount Analysis of Lymph Nodes and Pinna

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LNs and spleens were fixed in 10% buffered formalin and embedded in paraffin. Sections were cut at 5μm and stained with H&E. For immunohistochemistry, organs were frozen with OCT compound (Scigen Tissue-Plus, Gardena, CA). Frozen sections were cut at 6μm, fixed with cold acetone, blocked with 5% goat or donkey serum (Jackson ImmunoResearch, West Grove, PA) and incubated with the indicated antibodies (Key resources table). For pinna whole mounts, ears were depilated, peeled into 2 pieces, and fixed with 4% paraformaldehyde (PFA) for 15 minutes at room temperature. Samples were further processed as described previously (Brinkman et al., 2016 (link); Piao et al., 2018 (link)). LEC monolayers were stained as described previously (Xiong et al., 2017 (link)). Images were acquired with a Nikon Eclipse 700 (Nikon, Melville, NY, USA) and analyzed with Volocity image analysis software (Perkin Elmer, Waltham, MA). The positive staining area percentage was quantified based on at least three independent experiments with 3 mice/group, 3 LN/mouse, 3 sections/LN, 3–5 fields/section.

Saxena V., Piao W., Li L., Paluskievicz C., Xiong Y., Simon T., Lakhan R., Brinkman C.C., Walden S., Hippen K.L., WillsonShirkey M., Lee Y.S., Wagner C., Blazar B.R, & Bromberg J.S. (2022). Treg tissue stability depends on lymphotoxin beta-receptor- and adenosine-receptor-driven lymphatic endothelial cell responses. Cell reports, 39(3), 110727.

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Immunofluorescence Staining Protocol

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Cells were fixed with 4% paraformaldehyde at room temperature for 20 minutes and blocked with 10% goat or donkey serum (Jackson ImmunoResearch, West Grove, PA). For cytoplasmic protein staining, 0.1% Triton X was added for permeabilization. Primary antibodies were then added and incubated at 4°C overnight. Cells were washed and exposed to secondary antibodies conjugated with fluorochromes before co-staining with DAPI. All antibodies were listed in table S4.

Hong S.G., Winkler T., Wu C., Guo V., Pittaluga S., Nicolae A., Donahue R.E., Metzger M.E., Price S.D., Uchida N., Kuznetsov S.A., Kilts T., Li L., Robey P.G, & Dunbar C.E. (2014). Path to the clinic: Assessment of iPSC-based cell therapies in vivo in a non-human primate model. Cell reports, 7(4), 1298-1309.

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Immunohistochemistry and Whole Mount Analysis of Lymph Nodes and Pinna

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Immunofluorescence Imaging of BMMs

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BMMs were seeded onto 8-well glass chamber slides (Nunc) at 2×10⁴ cells/well. At each timepoint, BMMs were washed three times with PBS, then fixed with 4% paraformaldehyde (Electron Microscopy Sciences) in PBS for 10 minutes. BMMs were permeabilized with 0.25% Triton-X and blocked with 5% goat or donkey serum (Jackson Immunoresearch) and 5% BSA (Cell Signaling Technologies). Primary antibody incubation was done overnight at 4°C, followed by washing three times with PBS and incubation with Alexa-fluorophore conjugated secondary antibody (Life Technologies) for one hour in the dark. BMMs were then washed, counterstained with DAPI, mounted on coverslips, and imaged using a confocal microscope (Olympus) at 63X.

Maciag K., Raychowdhury R., Smith K., Schneider A.M., Coers J., Mumbach M.R., Schwartz S, & Hacohen N. (2021). IRF3 Inhibits IFNγ-mediated Restriction of Intracellular Pathogens in Macrophages Independently of IFNAR. Journal of leukocyte biology, 112(2), 257-271.

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Immunofluorescence Staining of Mouse Brain

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Brains from mice were harvested and placed in 4% PFA for 24 h. Following PFA fixation, brains were moved to a solution of 30% sucrose for 24 h, and were then embedded in OCT and flash frozen on dry ice. Samples were stored at −20 °C until cutting. After cutting, sections were blocked in 1× PBS containing 0.1% triton, 0.05% Tween 20, and 2% goat or donkey serum (Jackson ImmunoResearch) for 1 h at room temperature. Sections were then incubated with primary Abs overnight at 4 °C. Sections were then washed with PBS, and incubated with secondary Abs for 1 h at room temperature. Sections were then washed, and nuclear stained with DAPI (Thermo Fisher Scientific) for 5 min at room temperature. Finally, sections were mounted, covered in Aquamount (Lerner Laboratories), and covered with coverslips (Thermo Fisher Scientific). All images were captured using a Leica TCS SP8 Confocal microscopy system. Images were analyzed using either ImageJ or Imaris software.
Antibodies used include: pro-IL-1β (NJTEN3), iNOS (product # PA5-16855) 1:400, CD31 (390), VCAM-1 (429), ICAM-1 (YN1/1.7.4), c⁻Rel (G-7), IL-1α (ALF-161) 1:100, IL-1R1 (cat # AF771) 1:100, p65 (D14E12), Iba1 (cat # 019-19741) 1:1000, and laminin (product # CL54851AP-1). Dilutions are 1:200 unless otherwise noted.

Batista S.J., Still K.M., Johanson D., Thompson J.A., OʼBrien C.A., Lukens J.R, & Harris T.H. (2020). Gasdermin-D-dependent IL-1α release from microglia promotes protective immunity during chronic Toxoplasma gondii infection. Nature Communications, 11, 3687.

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Immunohistochemistry of Mouse Brain Sections

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Brains from mice were harvested and placed in 4% PFA for 24 hours. Following PFA fixation, brains were moved to a solution of 30% sucrose for 24 hours, and were then embedded in OCT and flash frozen on dry ice. Samples were stored at -20°C until cutting. After cutting, sections were blocked in 1X PBS containing 0.1% triton, 0.05% Tween 20, and 2% goat or donkey serum (Jackson ImmunoResearch) for 1 hour at room temperature. Sections were then incubated with primary Abs overnight at 4°C. Sections were then washed with PBS, and incubated with secondary Abs for 1 hour at room temperature. Sections were then washed, and nuclear stained with DAPI (Thermo Fisher Scientific) for 5 minutes at room temperature. Finally, sections were mounted, covered in Aquamount (Lerner Laboratories), and covered with coverslips (Thermo Fisher Scientific). All images were captured using a Leica TCS SP8 Confocal microscopy system. Images were analyzed using either ImageJ or Imaris software.

Batista S.J., Still K.M., Johanson D.M., Thompson J., O’Brien C.A., Lukens J.R., & Harris T.H. (2020). Gasdermin-D-dependent IL-1αrelease from microglia promotes protective immunity during chronicToxoplasma gondiiinfection.

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