Normal donkey serum (nds)

Manufactured by Abcam

Sourced in United Kingdom, United States, China

Normal donkey serum is a laboratory reagent derived from the blood serum of healthy donkeys. It is designed to be used as a blocking agent in various immunological techniques, such as immunohistochemistry, Western blotting, and ELISA, to reduce non-specific binding of antibodies.

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Lab products found in correlation

Triton x 100, by Merck Group (6 mentions) 4 6 diamidino 2 phenylindole (dapi), by Merck Group (5 mentions) 4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (4 mentions) Bovine serum albumin (bsa), by Merck Group (4 mentions) Tween 20, by Merck Group (4 mentions) Duolink pla technology kit, by Merck Group (3 mentions) Paraformaldehyde (pfa), by Merck Group (3 mentions) Triton x, by Merck Group (2 mentions) Bovine serum albumin (bsa), by Abcam (2 mentions) Normal donkey serum (nds), by Merck Group (2 mentions) Eclipse ti e inverted csu x1 spinning disk confocal microscope, by Nikon (2 mentions) Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions) Mouse anti g3bp, by BD (2 mentions) Dapi nuclear staining, by Thermo Fisher Scientific (2 mentions) Sodium citrate buffer, by Merck Group (2 mentions) Alexafluor 488 labeled donkey anti rabbit igg labeled, by Abcam (2 mentions) Tween 20, by Abcam (2 mentions) Lsm 510 meta confocal microscope, by Zeiss (2 mentions) Cm3050, by Leica (2 mentions) Triton x 100, by Thermo Fisher Scientific (2 mentions)

Spelling variants of this product

Donkey serum (50 citations) Normal serum (3 citations)

52 protocols using normal donkey serum (nds)

Autophagosome Localization and Analysis Protocol

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The experiment aimed to realize autophagosome localization and semi-quantitative analysis. 1) Cell slides (12-well plate) were prepared. 2) 4 % fresh paraformaldehyde was used to fix for 30 min, at 4 °C. 3) Using 0.5 % Triton X-100 (Beyotime, cat: P0096) treatment for 15 min to complete permeabilization. 4) Block with 10 % Normal Donkey Serum (Abcam, cat: ab7475) at rt for 30 min, then dilute the primary antibody with 1 % Normal Donkey Serum and incubate at 4 °C overnight. 5) Dilute the fluorescently labeled secondary antibody with 1 % Normal Donkey Serum, and incubate at rt for 1 h in the dark. 6) DAPI was used to stain the nucleus of IEC-6 cells. 7) Mount the slides with the ProLong™ Gold Antifade Mountants (cat: P10144), and then take pictures ( × 630) with the confocal laser scanning microscope (Zeiss, GER). Information on the antibodies used is recorded in Table 3.Table 3

Information on antibodies used in immunofluorescence.

Table 3

Antibody	Company	Cat	Dilution
LC3 A/B	CST	12741	1: 100
β-tubulin	CST	86298	1: 100
Donkey Anti-Mouse IgG H&L (Alexa Fluor® 647)	Abcam	ab150107	1: 400
Donkey Anti-Rabbit IgG H&L (Alexa Fluor® 488)	Abcam	ab150073	1: 400

Chen S., He R., Li Y, & Zhang S. (2024). Pure total flavonoids from Citrus ameliorate NSAIDs-induced intestinal mucosal injury via regulation of exosomal LncRNA H19 and protective autophagy. Heliyon, 10(9), e29797.

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Immunofluorescence Imaging of Immune Markers

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For the detection of EETs, PBEs were stained with EDN, CD63, and 4’,6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich) as previously described.32 (link) Cells were blocked with 5% BSA in 10% normal donkey serum (Abcam) and incubated overnight with appropriate primary antibodies.
For the detection of inflammatory levels in murine lung tissues, the fixed tissues were deparaffinated and rehydrated. The slides were blocked with 5% BSA in 10% normal donkey serum (Abcam) at room temperature for 1 hour and stained with MBP, CD63, and DAPI.29 (link)
After primary antibody incubation, cells/slides were stained with Alexa Flour 488-conjugated donkey anti-rabbit and 594-conjugated donkey anti-mouse (Thermo Fisher Scientific) for 1 hour. The slides were stained with DAPI (1:1,000) for 5 minutes. Fluorescent images were acquired using confocal laser scanning microscopy at the Three-Dimensional Immune System Imaging Core Facility (LSM710; Cal Zeiss Microscopy GmbH, Jena, Germany).

Cao T.B., Quoc Q.L., Yang E.M., Moon J.Y., Shin Y.S., Ryu M.S., Choi Y, & Park H.S. (2023). Tissue Inhibitor of Metalloproteinase-1 Enhances Eosinophilic Airway Inflammation in Severe Asthma. Allergy, Asthma & Immunology Research, 15(4), 451-472.

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Histological Analysis of Tissue Samples

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Hematoxylin and eosin (H&E) staining and immunohistochemical staining were described previously.^[²³^] Briefly, tissues were perfused with 0.9% NaCl, fixed in 4% paraformaldehyde overnight, and embedded in paraffin. The slices were dewaxed for H&E staining. For immunohistochemical staining, slices were repaired antigen in antigen repair buffer (Cat. C1032; Solarbio Science & Technology), blocked by Normal Donkey Serum (Cat. ab7475; Abcam), and incubated by primary and secondary antibodies. The images of all slices were taken by an inverted fluorescence microscope (Leica).

Wang D., Wu H., Yang J., Li M., Ling C., Gao Z., Lu H., Shen H, & Tang Y. (2022). Loss of SLC46A1 decreases tumor iron content in hepatocellular carcinoma. Hepatology Communications, 6(10), 2914-2924.

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Visualization of Intracranial Injections in Rats

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Isoflurane-anesthetized rats were intracranially injected via the same guide cannulae with 0.5 μL per side of diluted Normal Donkey Serum (Abcam) in 0.9% saline at 0.25 μL/min, followed by transcardial perfusion. Perfused brains were sliced, 30 μm coronally, and collected in 4 parallel series. For visualization of injection sites, one series of sectioned frontal cortex from each rat was processed using immunohistochemistry for donkey serum similar to procedures described in [49 (link)]. Briefly, slices were thoroughly washed and blocked for at least one hour in an incubation solution (PBS containing 0.1% bovine serum albumin and 0.4% triton-X-100). Then, slices were incubated with biotinylated goat anti-donkey secondary antibody (Thermo-Fisher) 1:500 dilution in incubation solution for one hour, followed by washing and exposure to avidin-biotin-horseradish peroxidase (ABC-Elite, 1:1000 dilution in PBS; Vector Laboratories). Complexes were visualized by a 15 min exposure to chromogen solution containing 0.02% 3,3’-diaminobenzidine tetrahydrochloride (DAB, Sigma). The reaction was terminated by extensive washing in PBS, then slices were mounted on cover-slipped glass slides and visualized under light microscope. All visualized injection sites were of expected size without excessive damage to the surrounding OFC tissue.

Race N.S., Andrews K.D., Lungwitz E.A., Alvarez S.M., Warner T.R., Acosta G., Cao J., Lu K.H., Liu Z., Dietrich A.D., Majumdar S., Shekhar A., Truitt W.A, & Shi R. (2021). Psychosocial impairment following mild blast-induced traumatic brain injury in rats. Behavioural brain research, 412, 113405.

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Quantification of Vascular Cell Interactions

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Third-order MAs and fourth-order PAs were isolated and pinned down en face on a Sylgard block. Arteries were fixed in 4% paraformaldehyde for 15 minutes, washed three times with PBS, and then incubated in a solution of 0.2% Triton X for 30 minutes at room temperature. Following this latter permeabilization step, arteries were blocked by incubating with either 5% normal donkey serum (Abcam plc) or 300 mM glycine at room temperature for 1 hour. Arteries were then washed three times with PBS and incubated overnight at 4°C with primary antibodies. The following day, the PLA protocol was performed as described by the manufacturer of the Duolink PLA Technology kit (Sigma-Aldrich, St. Louis, MO, USA). After incubating arteries with 0.3 μM DAPI nuclear stain (Invitrogen, Carlsbad, CA, USA) for 10 minutes at room temperature in the dark, PLA imaging and analysis were performed using an Andor Revolution spinning-disk confocal imaging system and Imaris 9.3 software (Bitplane AG, Zurich, Switzerland), respectively. Images were obtained along the z-axis at a slice thickness of 0.02 μm from the top surface of ECs to the bottom surface where they contact SMCs.

Ottolini M., Daneva Z., Chen Y.L., Cope E.L., Kasetti R.B., Zode G.S, & Sonkusare S.K. (2020). Mechanisms underlying selective coupling of endothelial Ca2+ signals with eNOS versus IK/SK channels in systemic and pulmonary arteries. The Journal of physiology, 598(17), 3577-3596.

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Immunofluorescence Staining for Tissue Integrity

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Immunofluorescence staining was performed after 7 days of drug treatment to investigate tissue integrity and presence of cell-specific markers. Membranes were excised from biochips and cells were fixed with ROTIHistofix 4% (Carl Roth) for 10 min at RT. Permeabilization and blocking was performed by adding PBS (Lonza) containing 0.1% saponin (Carl Roth) and 3% normal donkey serum (Abcam, Cambridge, UK) for 30 min at RT. The membrane was subsequently divided with scissors to independently stain the vascular and hepatic cell layers with the primary antibody solution. Primary antibodies ASGPR1 (BD Biosciences, Heidelberg, Germany), CYP3A4 (Sigma-Aldrich), α-GST (BIOZOL, Eching, Germany), CD32b (BIOZOL) and CD206 (Abcam) were incubated at 4 °C overnight. Membranes were washed with PBS/0.1% saponin and incubated with secondary antibodies DAPI (Thermo Fisher Scientific), donkey-anti-mouse-AF647 (Thermo Fisher Scientific), donkey-anti-rabbit-Cy3 (Jackson ImmunoResearch, Cambridgeshire, UK) and donkey-anti-goat-AF488 (Thermo Fisher Scientific) for 1 h at RT. Stained membranes were washed twice with PBS/0.1% saponin, once with PBS and lastly with AQUA AD iniectabilia (B. Braun, Melsungen, Germany). Samples were embedded in fluorescent mounting medium (Agilent Technologies, Waldbronn, Germany).

Kaden T., Graf K., Rennert K., Li R., Mosig A.S, & Raasch M. (2023). Evaluation of drug-induced liver toxicity of trovafloxacin and levofloxacin in a human microphysiological liver model. Scientific Reports, 13, 13338.

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Neuronal Identification and Characterization

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We used NeuroFluor NeuO (Stemcell Technologies, Vancouver, Canada) live staining to locate neurons on the array before the stimulation experiments. The cells were incubated for 1 h at 37°C with 2 mL growth medium containing 0.15% NeuO. The chips were then washed 2 times with growth medium.
We also performed neuron fixation after stimulation experiments by using 4% paraformaldehyde (Life Technologies, Thermo Fisher Scientific). Sample permeabilization and blocking of non-specific antibody binding were done using a PBS 1× solution containing: 10% normal donkey serum (NDS) (Sigma-Aldrich), 1% bovine serum albumin (BSA) (Sigma-Aldrich), 0.02% Na-Az (Sigma-Aldrich), 0.5% Triton X (Sigma-Aldrich). Primary and secondary antibodies were diluted in a PBS 1× solution containing: 3% normal donkey serum (NDS), 1% bovine serum albumin (BSA), 0.02% Na-Az, 0.5% Triton X. We used antibodies against MAP2 (Abcam, Cambridge, United Kingdom), Ankyrin G (Santa Cruz Biotechnology, Dallas, TX, United States), and the fluorescent dye Hoechst (Invitrogen) to stain neurons, axonal initial segments (AIS), and nuclei. We imaged cells on the HD-MEA chip with a Nikon NiE upright confocal microscope, with a Yokogawa W1 spinning disk scan head, 6 laser lines and a fluorescence recovery after photobleaching (FRAP) unit.

Ronchi S., Fiscella M., Marchetti C., Viswam V., Müller J., Frey U, & Hierlemann A. (2019). Single-Cell Electrical Stimulation Using CMOS-Based High-Density Microelectrode Arrays. Frontiers in Neuroscience, 13, 208.

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Immunohistochemistry of Paraffin Sections

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Paraffin embedded tissue sections (10 μm) were deparaffinized with xylene and rehydrated using a decreasing gradient of ethanol. Heat-induced antigen retrieval was performed using citrate buffer (10 mM Citric acid, 0.05% Tween-20, pH 6.0) for 20 min at 90–95°C. To reduce non-specific binding, blocking was carried out with 10% Normal donkey serum (EMD Millipore, Burlington, Massachusetts) and 0.2% Triton-X100 in PBS for 1 h at room temperature. A list of all the primary and secondary antibodies used are listed in Supplementary Table 3. Primary antibodies were prepared in antibody diluent solution (2.5% Normal donkey serum, 0.25% sodium azide, 0.2% trition X 100, and PBS, Abcam, Cambridge, Massachusetts), and sections were covered with primary antibodies overnight at 4°C. Next, each well was washed with PBS five times and then incubated with the respective secondary antibodies for 1 h, followed by mounting with VECTASHIELD® antifade mounting medium containing DAPI (Vector Labs, Burlingame, California) on glass sides. Slides were dried overnight and then imaged using a Nikon Eclipse C1 microscope.

Massey N., Shrestha D., Bhat S.M., Padhi P., Wang C., Karriker L.A., Smith J.D., Kanthasamy A.G, & Charavaryamath C. (2022). Mitoapocynin Attenuates Organic Dust Exposure-Induced Neuroinflammation and Sensory-Motor Deficits in a Mouse Model. Frontiers in Cellular Neuroscience, 16, 817046.

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Hepatitis B Virus Immunofluorescence Assay

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Following HBV inoculation as described above, cells were washed twice with cold PBS, followed by incubation with 4% paraformaldehyde (PFA)/PBS for 30 min at room temperature for immobilization. Cells were then treated with 0.25% Triton-X100/PBS for 5 min at room temperature following 3 washes with PBS, then blocking with 2% bovine serum albumin (BSA) and 2.5% normal donkey serum (Abcam)/PBS. Cells were incubated with anti-HBsAg goat IgG polyclonal antibody (bs-1557G, 1:1000) (Bioss, Boston, MA) as a primary antibody overnight at 4 °C. After washing, cells were incubated with Alexa Fluor 488-labeled donkey anti-goat IgG (H + L) (A-11055, 1:1000) (Life Technologies) for 1 h. Cells were then stained with 4’,6-diamidino-2-phenylindole (DAPI) for 1 h and observed under a fluorescence microscope.

Sakurai F., Mitani S., Yamamoto T., Takayama K., Tachibana M., Watashi K., Wakita T., Iijima S., Tanaka Y, & Mizuguchi H. (2017). Human induced-pluripotent stem cell-derived hepatocyte-like cells as an in vitro model of human hepatitis B virus infection. Scientific Reports, 7, 45698.

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Immunohistochemical Analysis of Brain Sections

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Post‐fixed brain hemispheres were sliced at the LEICA VT1000S vibrating microtome to obtain 50 µm thick sagittal sections. The brain sections were put into separate wells of a 24‐well plate and rinsed in a solution of 50 mM Tris‐Cl, 150 mM NaCl (TBS, pH 7.6) for 10 min. Permeabilization and blocking of non‐specific binding sites was achieved by incubation in 500 μl of blocking solution (10% [v/v] Normal Donkey Serum, Abcam; 1% [v/v] Triton‐X100, Sigma‐Aldrich; TBS) for 2 h at room temperature. The brain sections were then incubated in 500 μl of primary antibody solution overnight at 4°C. On the following day, the brain sections were rinsed six times in TBS. Each washing step was carried out for 10 min. The sections were then incubated for 2 h, at room temperature, in 500 μl of secondary antibody solution. Finally, the sections were rinsed six times with TBS and mounted on SuperFrost Plus Adhesion glass slides (Thermo Fisher Scientific) with Fluromount‐G without addition of DAPI (Thermo Fisher Scientific), unless otherwise specified. The glass slides were stored at 4°C, protected from light to prevent photobleaching.

Marino M., Zhou L., Rincon M.Y., Callaerts‐Vegh Z., Verhaert J., Wahis J., Creemers E., Yshii L., Wierda K., Saito T., Marneffe C., Voytyuk I., Wouters Y., Dewilde M., Duqué S.I., Vincke C., Levites Y., Golde T.E., Saido T.C., Muyldermans S., Liston A., De Strooper B, & Holt M.G. (2022). AAV‐mediated delivery of an anti‐BACE1 VHH alleviates pathology in an Alzheimer's disease model. EMBO Molecular Medicine, 14(4), e09824.

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