Goat serum

Manufactured by Merck Group

Sourced in United States, United Kingdom, Germany, Spain, France, Italy, Japan, China, Canada

Goat serum is a biological material derived from the blood of healthy goats. It is a complex mixture of proteins, hormones, and other biomolecules. Goat serum is commonly used as a supplement in cell culture media to support the growth and maintenance of various cell types in laboratory settings.

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719 protocols using goat serum

Immunohistochemical Analysis of Paraffin-Embedded Tissues

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Paraffin-embedded tissue samples were cut into 5 µm sections, deparaffinized and rehydrated. For antigen retrieval, slides were boiled in citrate buffer (0.01 M, pH 6.0) using a microwave oven on high power for 5 min and cooled down to room temperature. After incubation in 3% aqueous H₂O₂ to quench the endogenous peroxidase, the sections were washed in PBST (PBS with 0.1% Tween-20, v/v) washing buffer, blocked with 5% goat serum (EMD Millipore) diluted in PBST at room temperature for 1 h and incubated with primary antibodies diluted in 2% goat serum in PBST at 4 °C overnight.
For immunohistochemistry, slides were incubated with appropriate biotinylated secondary antibodies (Supplementary Table 3) for 1 h, and then processed according to the ABC Peroxidase Standard Staining Kit (Thermo Fisher Scientific) for 30 min. The slides were stained with 3,3′-diaminobenzidine (Abcam) for 5 s to 5 min and counterstained with haematoxylin (Thermo Fisher Scientific) for 45 s. The images were scanned using the Leica Aperio AT2 system at Stanford Human Pathology/Histology Service Center. Serial sections were incubated with GS and MYC-tag primary antibodies, and a cluster of cells (at least 20 cells) positive both for GS and MYC-tag were counted as early foci. The antibodies used in this study are shown in Supplementary Table 3.

Fan W., Adebowale K., Váncza L., Li Y., Rabbi M.F., Kunimoto K., Chen D., Mozes G., Chiu D.K., Li Y., Tao J., Wei Y., Adeniji N., Brunsing R.L., Dhanasekaran R., Singhi A., Geller D., Lo S.H., Hodgson L., Engleman E.G., Charville G.W., Charu V., Monga S.P., Kim T., Wells R.G., Chaudhuri O, & Török N.J. (2024). Matrix viscoelasticity promotes liver cancer progression in the pre-cirrhotic liver. Nature, 626(7999), 635-642.

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Immunofluorescence Staining of Cryo-Sectioned Skin

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For immunofluorescence staining of cryo-sections, dorsal skin was embedded in OCT (Sakura, fisher scientific, Schwerte, Germany), immediately frozen, cut into 5-µm sections, and post-fixed with 4% PFA (Roth, Krems, Austria) for 30 min at room temperature. Subsequently, skin sections or epidermal sheets were blocked with 5% goat serum (Merck, Darmstadt, Germany) and 2% BSA TBS-T (Merck, Darmstadt, Germany) for 1 h and incubated with primary antibodies diluted in 5% goat serum and 2% BSA TBS-T at 4 °C overnight. Subsequently, slides were rinsed and incubated with an appropriate secondary antibody and Hoechst (Sigma-Aldrich, St. Louis, MO, UAS) for 2 h in a dark humidified slide chamber. Tissue sections were mounted, and pictures were taken using a Nikon eclipse 80i microscope (Nikon, Tokyo, Japan). Antibodies used are listed in Table 1.

Bauer T., Gubi D., Klufa J., Novoszel P., Holcmann M, & Sibilia M. (2021). Ex-Vivo Skin Explant Culture Is a Model for TSLP-Mediated Skin Barrier Immunity. Life, 11(11), 1237.

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Quantifying DNA Double-Strand Breaks

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Post irradiation cells were fixed and stained for γH2AX foci to evaluate DNA DSB formation and DAPI for nuclei masking. Briefly, cells were washed with PBS and fixed 1hr post irradiation with an ice cold solution consisting of 95% Ethanol (Chem-Supply) and 5% Acetic acid (Chem-Supply) for 10 mins. Following fixation cells were permeabilised for 15 mins using a PBS solution containing 0.5% Triton X-100 and then blocked using a buffer solution consisting of 5% Goat serum (Sigma-Aldrich) in PBS for 1 hr in a humidified incubator at 37°and 5% CO₂. After blocking cells were incubated for a further 1 hour in a humidified incubator at 37° and 5% CO₂ with 1/500 mouse anti-γH2AX (Millipore) antibody in PBS + 1% Goat serum. Fluorescent secondary antibody staining was performed by incubating the cells with Goat anti-mouse Alexa 488 (Abcam) at a 1/500 dilution in 1% Goat serum for 1 hr in the same conditions as the primary antibody step. Cells were then stained for nuclei identification and DNA content analysis using a DAPI solution (1 μg/ml) (Sigma-Aldrich) for 15 mins at room temperature. Finally cells were washed with MQ water for imaging.

Turnbull T., Douglass M., Williamson N.H., Howard D., Bhardwaj R., Lawrence M., Paterson D.J., Bezak E., Thierry B, & Kempson I.M. (2019). Cross-Correlative Single-Cell Analysis Reveals Biological Mechanisms of Nanoparticle Radiosensitization. ACS nano, 13(5), 5077-5090.

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Immunostaining of Myofibres and MPCs

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For immunostaining, either myofibres or MPCs were permeabilised in PBS 0.5% Triton X-100 (PBST) for 15 min, blocked in Goat Serum 5% (Sigma Aldrich) in PBST and incubated with primary antibodies at indicated concentrations overnight in Goat Serum 2% in either PBST (0.1% TritonX) (myofibres) or PBS (MPCs). Primary antibodies used were against: phospho-S6 ribosomal protein (Ser240/244), (1:1000; #5364; Cell Signaling), GFP (13970 (1:400), Abcam), Myosin (A4.1025 (1:5) (Blagden et al., 1997)

Ganassi M., Badodi S., Wanders K., Zammit P.S., & Hughes S.M. (2020). Myogenin is an Essential Regulator of Adult Myofibre Growth and Muscle Stem Cell Homeostasis.

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Cryosection Staining for Alzheimer's Markers

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Cryosections were dried in the hood for 1 h, followed by 5 min incubation in 70% ethanol, 2 min in distilled H2O and stained with Amylo-Glo dye [56] in saline solution for 5 min, then washed in distilled H2O for 15 s and in TBS (50 mM TBS, pH 8.0), 3×5 min. Samples were then permeabilized in TBS-T (50 mM TBS, pH 8.0, 0.5% Triton X-100, Sigma-Aldrich, St. Louis, MO, USA) for 30 min, blocked in 5% goat serum (Sigma-Aldrich, St. Louis, MO, USA) in TBS-T for 1 h and incubated with primary antibodies diluted in 5% goat serum in TBS-T overnight. Antibodies used were: APP (C-terminal antibody, Epitomics), BACE-1 (Epitomics), LAMP1 (Santa Cruz), Transferrin Receptor (TfR, Life Technologies), neurofilament (NFL, Sigma-Aldrich), Sez6 and Sez6L (N-terminal antibodies, gift Dr. Lichtenthaler). Next day, sections were incubated with secondary antibody goat anti-rabbit-Alexa488, anti-mouse-Alexa594 and/or anti-rat-Alexa647 (Molecular Probes, Invitrogen, Waltham, MA, USA) for 3 h and mounted (Fluoromount, Sigma-Aldrich, St. Louis, MO, USA). Confocal images were acquired on an inverted laser scanning confocal microscope Leica TCS SP8 with the software LAS X (Leica, Wetzlar, Germany), and additional image processing and quantification were performed using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

Dominko K., Rastija A., Smiljanic K., Mladenovic A., Lešnjaković L., Kanazir S., Milanovic D, & Hecimovic S. (2022). Amyloid-ß plaque formation and BACE1 accumulation in the brains of a 5xFAD Alzheimer's disease mouse model is associated with altered distribution and not proteolysis of BACE1 substrates Sez6 and Sez6L. Mechanisms of ageing and development, 207.

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Immunocytochemistry of Cardiomyocytes

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Cell sheets differentiated for 34 days were dissociated into single cells with TryPLE and seeded at low density onto LN-521+LN-221coated plates. After 3 days, CMs were washed once with PBS and fixed with 4% paraformaldehyde (PFA) (Sigma, Cat.#28908) for 20 mins at 4 C. The cells were then washed with PBS and blocked with blocking buffer containing 1% BSA, 5% goat serum (Sigma, Cat.#G9023) and 0.1% Triton X (Sigma, Cat.#T8787) for 1 hr at room temperature. After blocking, cells were incubated with primary antibody: TNNI3 (Santa cruz, Cat.#sc-15368, 1:20), NKX2-5 (Santa Cruz, Cat.#sc-14033, 1:100), TNNT2 (1:200), a-actinin (Sigma, Cat.#A7811, 1:500) in PBS containing 1% BSA and 5% goat serum at room temperature for 1 hour. Following this, antibody solution was removed and cells washed thrice with PBS on a shaker for 5 mins each. Subsequently, the cells were incubated at room temperature with Alexa-conjugated secondary antibody (ThermoFisher, 1:1000) on a shaker at low speed in the dark for 1 hr. Finally, cells were washed with PBS twice (5 mins) and ProLongâ Gold Antifade Mountant with DAPI (ThermoFisher, Cat.#P36931) added to stain the nuclei. Cells were visualized immediately in Leica TCS sp8 confocal fluorescence microscope.

Yap L., Wang J.W., Moreno-Moral A., Chong L.Y., Sun Y., Harmston N., Wang X., Chong S.Y., Vanezis K., Öhman M.K., Wei H., Bunte R., Gosh S., Cook S., Hovatta O., de Kleijn D.P.V., Petretto E, & Tryggvason K. (2019). In Vivo Generation of Post-infarct Human Cardiac Muscle by Laminin-Promoted Cardiovascular Progenitors. Cell reports, 26(12).

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Quantification of Autoantibody Binding

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Nunc MaxiSorp 96-well plates (Fisher Scientific, Richardson, TX, USA) were coated with 2.0 μg/mL of the recombinant KRT1-WT, KRT1-MU, or KRT1-DEL in 0.05 M carbonate/bicarbonate buffer (pH 9.6) by incubation at 4°C overnight. Plates were coated with 0.48 μg/mL of His-Tagged Human ATG8/GABARAPL1 protein (Sino Biological Inc., Beijing, CHN) as controls. The coated plates were blocked by incubation with 10% goat serum (Sigma-Aldrich) in PBS at 37°C for 2 h. The plates were then incubated with test serum diluted 1 : 50 in an incubation solution of 10% goat serum in PBS at 4°C overnight. After five washes with PBS, HRP-conjugated goat anti-human antibodies (Jackson Laboratories) diluted 1 : 5,000 were added, and samples were incubated at 37°C for 2 h. After five washes with PBS, 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonate) (ABTS, Shanghai Yuanye Biotechnology Co., Shanghai, CHN) was added to the plates, and the reaction was stopped after 30 minutes using 2 M sulfuric acid. Optical density was read using a Stat Fax-2100 microplate reader (Awareness Technology, Inc., Palm City, FL, USA) at 405 nm. Each sample was tested in triplicate, and a median value, from which the goat serum control value was subtracted, was used for analysis. Experiments on each sample were repeated to ensure reproducibility.

Guo X., Hu J., Luo W., Luo Q., Guo J., Tian F., Ming Y, & Zou Y. (2017). Analysis of Sera of Recipients with Allograft Rejection Indicates That Keratin 1 Is the Target of Anti-Endothelial Antibodies. Journal of Immunology Research, 2017, 8679841.

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Immunostaining of BM-LMCs with Anti-adiponectin

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Anti-adiponectin antibody (Abcam, USA; ab22554) was used for immunostaining of BM-LMCs with a 1/400 dilution. The wells were firstly washed with PBS and then incubated with 4% of cold paraformaldehyde for 10 followed by 5 min at RT. Triton X-100 0.25% was added for 20 min at RT. After washing with PBS (Gibco), H₂O₂ 0.3% was added for 20 min in a dark place at RT followed by two times washing with PBS. The cells were incubated with 5% goat serum (Sigma) for 45 min at RT. After removing goat serum, the first antibody diluted in bovine serum albumin (BSA) (Sigma)/PBS 0.1% was added and the plate was kept at 4 °C overnight. Then, the wells were washed with PBS–Tween 0.1%, 3 × 5 min. After incubation with BSA/PBS 1% for 30 min at RT, the second antibody (Abcam, ab205719, 1/1000) was added to wells for 3 h. Subsequently, the wells were washed with PBS–Tween 0.1%, 3 × 5 min. 3,3'-Diaminobenzidine (DAB) (Sigma, Germany) solution was used as chromogen on plates for 10 min at RT. The wells were washed with PBS, 2 × 5 min. Finally, PBS was added to the wells.

Moein S., Ahmadbeigi N., Adibi R., Kamali S., Moradzadeh K., Nematollahi P., Nardi N.B, & Gheisari Y. (2023). Regenerative potential of multinucleated cells: bone marrow adiponectin-positive multinucleated cells take the lead. Stem Cell Research & Therapy, 14, 173.

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Immunofluorescence Staining Protocol

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Portions of this section were published previously^{50 (link)}. For immunofluorescence staining, cells were grown on acid-washed 22 × 22 mm square no. 1½ glass coverslips (Corning; 2850–22). Cells on coverslips were washed three times in phosphate-buffered saline (PBS) for 5 min per wash, then fixed using 4% paraformaldehyde for 15 min. Cells on coverslips were then washed three times in PBS for 5 min per wash and permeabilized with 0.1% Triton X-100 for 10 min. After three more 5-min PBS washes, cells were blocked in 10% goat serum (Sigma; G9023) and 0.3 M glycine in PBS for 1 h. Three 5-min washes in PBS were repeated and cells were stained overnight in primary antibody solution (2% goat serum in PBS with appropriate primary antibodies). After three more 5-min PBS washes, cells were incubated with secondary antibody solution (2% goat serum in PBS with appropriate secondary antibodies) for 75 min. Hoechst 33342 stain (Sigma; 14533) was added to the secondary antibody solution at a concentration of 10 µg/mL in some assays to image DNA. Coverslips were then washed three times in PBS for 5 min per wash, briefly washed twice with dH₂O, mounted on microscope slides using VECTASHIELD antifade mounting medium (Vector Laboratories; H-1000), and sealed with nail polish.

Wesley C.C., North D.V, & Levy D.L. (2024). Protein kinase C activity modulates nuclear Lamin A/C dynamics in HeLa cells. Scientific Reports, 14, 6388.

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Immunocytochemistry of hiPSC-Derived Cardiomyocytes

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For immunostaining, hiPSC-CMs were plated on thin black bottom 96 well plates (Corning). Cells were washed using PBS followed by fixing using 5% PFA (Sigma-Aldrich) for 15 min followed by 2x PBS washes. Fixed cells were then washed for 15 min in 0.1% Triton X-100 (Sigma-Aldrich, Gillingham, UK) in PBS followed by 3x further washes in PBS. Fixed cells were then blocked in 4% Goat Serum (Sigma-Aldrich, Gillingham, UK) in PBS for 1 h followed by a further 5 min PBS wash. Primary antibody was added to the plates, diluted in 4% Goat Serum in PBS (Alpha Actinin 1:800, Abcam) and left overnight at 4 °C on a rocking plate. The following day fixed cells were washed with 0.1% Tween (Sigma-Aldrich, Gillingham, UK) in PBS (3 × 10 min) followed by secondary antibody staining [Anti-Mouse Alexa Fluor^® 488 (Abcam, Cambridge, UK) diluted 1:500 in 4% Goat Serum (Sigma-Aldrich, Gillingham, UK) in PBS] for 1 h at room temperature. The fixed cells were then washed 3 times with 0.1% Tween-PBS (Sigma-Aldrich, Gillingham, UK) followed by incubation in PBS supplemented with DAPI (Tocris, Abingdon, UK) at 1:500 for 10 min. A further wash was performed using PBS after which the fixed cells were submerged in PBS and the plate wrapped in foil prior to imaging using the EVOS M5000 imaging system.

Johnson B.B., Reinhold J., Holmes T.L., Moore J.A., Cowell V., Bernardo A.S., Rushworth S.A., Vassiliou V, & Smith J.G. (2021). Modelling Metabolic Shifts during Cardiomyocyte Differentiation, Iron Deficiency and Transferrin Rescue Using Human Pluripotent Stem Cells. Metabolites, 12(1), 9.

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