Donkey serum

Manufactured by Dianova

Sourced in Germany

Donkey serum is a biological reagent derived from the blood of donkeys. It contains a variety of proteins, antibodies, and other biomolecules that can be used in various laboratory applications.

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Lab products found in correlation

Fluoromount g, by Calibre Scientific (1 mentions) Axio imager m2, by Zeiss (1 mentions) 4 6 diamidino 2 phenylindole (dapi), by Roche (1 mentions) Ab18197, by BD (1 mentions) Ab5603, by BD (1 mentions) Mouse anti map2, by Merck Group (1 mentions) Z0334, by Merck Group (1 mentions) Hoechst 33342 nuclear dye, by Thermo Fisher Scientific (1 mentions) Mouse anti nestin, by BD (1 mentions) Triton x 100, by Merck Group (1 mentions) Tx 100, by Merck Group (1 mentions) Dako fluorescence mounting medium, by Agilent Technologies (1 mentions) Fitc conjugated wga, by Merck Group (1 mentions) Vectashield dapi, by Vector Laboratories (1 mentions) Extravidin peroxidase, by Merck Group (1 mentions) Biotinylated secondary antibody, by GE Healthcare (1 mentions) 3 3 diaminobenzidine tetrahydrochloride dab, by Carl Roth (1 mentions) Histokitt, by Carl Roth (1 mentions) Anti pdx1, by R&D Systems (1 mentions) Ix81 microscope, by Olympus (1 mentions)

8 protocols using donkey serum

Immunocytochemistry Staining Protocol

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ICC was essentially performed as described previously [76] :
After rinsing of cells, fixation was done for 10 min at room temperature (RT) with 4% paraformaldehyde (PFA) in PBS (ThermoFisher Scientific). Subsequently, cells were washed with PBS and incubated with PBS containing 0.4% TritonX-100 and 5% donkey serum (DS, dianova, Hamburg, Germany) for 30 min at RT to permeabilize cells and block unspecific binding of antibodies. Primary Ab were diluted in Ab solution (1% DS and 0.1% TritonX-100 in PBS) and incubated overnight at 4°C. After extensive washing, secondary antibodies coupled to fluorophores were applied for 2 h at RT. After rinsing thrice with PBS, coverslips were mounted in SlowFade Gold Antifade Medium with 4',6-Diamidino-2-Phenylindole (DAPI, ThermoFisher Scientific).

Sondermann J.R., Barry A.M., Jahn O., Michel N., Abdelaziz R., Kügler S., Gomez-Varela D, & Schmidt M. (2019). Vti1b promotes TRPV1 sensitization during inflammatory pain. Pain, 160(2).

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Immunofluorescence Staining of PTX3 in Mouse Hearts

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Mouse hearts were prepared as described above. For antigen retrieval, deparaffinized sections were boiled for 60 min in 10 mm citrate buffer (10 mm sodium citrate, 0.05% Tween-20, pH 6.0) using a steamer. Afterward, sections were blocked for 60 min in PBS containing donkey serum (1.2 mg/ml; Dianova) and then with 1% Sudan black in 70% ethanol for 20 min in order to decrease autofluorescence of the tissue. After rigorous washing, sections were incubated with anti-PTX3 (1:100; Abcam, Cambridge, UK) primary antibody overnight at 4 °C. Sections were then washed again, and secondary donkey anti-rabbit IgG Alexa 633 labeled antibody (1:200; Invitrogen, Darmstadt, Germany) was applied for 60 min. Nuclei were counterstained by 0.4 μg/ml 4′6-diamidino-2′-phenylindole (DAPI) (Roche, Munich, Germany) in PBS for 10 min, and slides were mounted in Fluoromount G (Biozol, Munich, Germany). Images were taken on a fluorescence light microscope equipped with a digital camera (Axio Imager M2, Zeiss, Jena, Germany).

Cuello F., Shankar-Hari M., Mayr U., Yin X., Marshall M., Suna G., Willeit P., Langley S.R., Jayawardhana T., Zeller T., Terblanche M., Shah A.M, & Mayr M. (2014). Redox State of Pentraxin 3 as a Novel Biomarker for Resolution of Inflammation and Survival in Sepsis. Molecular & Cellular Proteomics : MCP, 13(10), 2545-2557.

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Immunocytochemistry of 2D NPC Cultures

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2D NPC cultures and cells on microcarriers were fixed with 4 % PFA in PBS, permeabilized with 0.1 % Triton-X100 (Sigma) for 15 min, blocked with 10 % donkey serum (Dianova, Ref. 017-000-121) for 30 min. Subsequently, cells were incubated with either rabbit anti-Sox2 (Millipore, AB5603, 1:400) and mouse anti-nestin (BD Biosciences, 611658, 1:250), rabbit anti-PCNA (abcam, ab18197, 1:1000) and mouse anti-nestin (BD Biosciences, 611658, 1:250) or rabbit anti-GFAP (Dako, Z0334, 1:250) and mouse anti-Map2 (Sigma, M1406 in. 1:1000) in blocking solution at 4 °C over-night. After washing with 1X DPBS three times, secondary antibody incubation was performed with donkey anti-rabbit Alexa Fluor568 IgG (H + L) (Life technologies, A10042, 1:500) and donkey anti-mouse Alexa Fluor488 IgG (H + L) (Dianova, 715-545-151, 1:500) in blocking solution for 3 h at room temperature in the dark. Unbound secondary antibody was washed away with 1X DPBS. Thereafter, cells were counterstained with Hoechst 33342 nuclear dye (Molecular Probes, Invitrogen, 1:1000 in DPBS) for 10 min and again washed with 1X DPBS.

Newland B., Ehret F., Hoppe F., Eigel D., Pette D., Newland H., Welzel P.B., Kempermann G, & Werner C. (2020). Static and dynamic 3D culture of neural precursor cells on macroporous cryogel microcarriers. MethodsX, 7, 100805.

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Quantifying Cardiomyocyte Nuclei by Immunolabeling

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For estimation of the mean number of nuclei per cardiomyocyte, 25-lm-thick paraffin sections of the left ventricle were immunolabeled with an N-cadherin antibody, FITC-labeled wheat germ agglutinin (WGA) and TO-PRO â -3 iodide to visualize cell borders of cardiac myocytes and nuclei. In short, tissue was deparaffinized in xylol and decreasing isopropanol concentrations. Sections were incubated with Dako Retrieval buffer pH 6.0 (Dako, Glostrup, Denmark) at 800 W for 20 min and, after washing steps, with 10% donkey serum (Dianova, Hamburg, Germany) and 0.15% TX-100 (Sigma-Aldrich, Steinheim, Germany) in PBS for 45 min at room temperature. Then, sections were incubated with a monoclonal mouse anti-pan cadherin antibody (Sigma-Aldrich, Steinheim, Germany) diluted 1 : 1000 in PBS containing 1% bovine serum albumin (BSA) and 0.15% TX-100 for 20 h at 4 °C. After subsequent washing, sections were incubated for 48 h at 4 °C with PBS/1% BSA/0.15% TX-100 containing Alexa488-linked goat-anti-mouse antibody (diluted 1 : 1000; Life Technologies, Carlsbad, CA, USA), FITC-conjugated WGA (diluted 1 : 100; Sigma-Aldrich, Steinheim, Germany) and TO-PRO â -3 iodide (diluted 1 : 750; Life Technologies, Carlsbad, CA, USA). Sections were embedded in Dako Fluorescence Mounting Medium and sealed with a cover slip.

Schipke J., Grimm C., Arnstein G., Kockskämper J., Sedej S, & Mühlfeld C. (2016). Cardiomyocyte loss is not required for the progression of left ventricular hypertrophy induced by pressure overload in female mice. Journal of anatomy, 229(1).

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Immune and Vascular Cell Imaging

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Cryosections (6 μm) were blocked with 10% donkey serum (Dianova) and stained with primary antibodies against Gr-1 and an endothelial specific lectin (LEA-1).

Retzlaff J., Thamm K., Ghosh C.C., Ziegler W., Haller H., Parikh S.M, & David S. (2017). Flunarizine suppresses endothelial Angiopoietin-2 in a calcium - dependent fashion in sepsis. Scientific Reports, 7, 44113.

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Immunohistochemical Staining of ICAM-1

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Ice-cold acetone-fixed cryosections (6 μm) were blocked with 10% donkey serum (Dianova) and stained with primary antibodies against ICAM-1. Paraformaldehyde-fixed (Merck, Darmstadt, Germany) and paraffin-embedded tissue sections (1.5 μm) were dehydrated and rehydrated with ascending and descending ethanol series including deparaffinising with Histoclear (Biozym, Hessisch Oldendorf, Germany). After blocking with 10% donkey serum, paraffin sections were stained with primary and a secondary antibody. Mounting was accomplished with VectaShield DAPI (Vector Laboratories Inc., Burlingame, CA).

Thamm K., Njau F., Van Slyke P., Dumont D.J., Park J.K., Haller H, & David S. (2016). Pharmacological Tie2 activation in kidney transplantation. World Journal of Transplantation, 6(3), 573-582.

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Immunohistochemical Staining Protocol

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A detailed protocol is provided in the supplementary methods. Briefly, 5 µm tissue sections were de-paraffinized in xylene and rehydrated using decreasing alcohol concentrations. Antigen retrieval and endogenous peroxidase block were performed in citric acid buffer (10 mM citric acid, pH 6, 0.1% Tween 20) and 3% H₂O₂ in PBS, respectively. Samples were then incubated in blocking solution (5% bovine serum albumin (BSA, Merk) and 1% donkey serum (Dianova GmbH) in PBS). Primary and secondary antibodies (see supplementary Table S3-4) were diluted in blocking solution and incubated in a dark humidified chamber. Biotinylated secondary antibodies (GE Healthcare, see supplementary Table S4) and ExtrAvidin-Peroxidase (Sigma-Aldrich) were diluted in PBS, and samples were incubated in a dark humid chamber. Staining was developed using 3,3′-diaminobenzidine-tetrahydrochloride (DAB; Roth) and counterstained using hematoxylin. Slides were dehydrated following the reverse order of the alcohol gradient and mounted with Histokitt (Carl Roth GmbH).

Prokakis E., Dyas A., Grün R., Fritzsche S., Bedi U., Kazerouni Z.B., Kosinsky R.L., Johnsen S.A, & Wegwitz F. (2021). USP22 promotes HER2-driven mammary carcinoma aggressiveness by suppressing the unfolded protein response. Oncogene, 40(23), 4004-4018.

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Immunocytochemistry of Differentiated hESCs

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For immunocytochemistry, hESCs were seeded on Matrigel-coated glass slides (SPL Life Sciences) and subjected to differentiation. The cells at day 8 (d8) were fixed in 4% (w/v) paraformaldehyde for 10–20 min at 4 °C and subsequently blocked for 20 min in PBS plus 0.2% Triton X-100 with 5% donkey serum (Dianova, Hamburg, Germany) and 1 mg/mL NaBH₄. Primary and secondary antibodies were diluted in PBS with 0.1% Triton X-100 and 0.1% donkey serum. Primary antibodies were incubated overnight at 4 °C. Secondary antibodies were diluted 1:500 and incubated for 1 h at room temperature. The primary antibodies anti-HNF1B (SantaCruz, sc-22840, Santa Cruz, CA, USA) and anti-PDX1 (R&D systems, AF2419, Minneapolis, MN, USA) were used. Secondary antibodies were obtained from Dianova (Hamburg, Germany) conjugated with AlexaFluor or Cy fluorophores. Finally, the slides were mounted with Immunoselect antifading mounting medium containing DAPI (Dianova). Stained cells were examined using an Olympus IX81 microscope (Olympus, Tokyo, Japan). For PDX1-positive cell counting, between 6–10 pictures for each condition from 3 experiments were taken and the cells were quantified using the plugin Image-based Tool for Counting Nuclei (ITCN) on Image J.

Dettmer R., Cirksena K., Münchhoff J., Kresse J., Diekmann U., Niwolik I., Buettner F.F, & Naujok O. (2020). FGF2 Inhibits Early Pancreatic Lineage Specification during Differentiation of Human Embryonic Stem Cells. Cells, 9(9), 1927.

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