Normal goat serum (ngs)

Manufactured by Cell Signaling Technology

Sourced in United States

Normal goat serum is a laboratory reagent used to block non-specific binding in immunoassays and immunohistochemistry applications. It is derived from the serum of healthy goats and contains a mixture of proteins, including immunoglobulins, which can help reduce the background signal in these experiments.

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Lab products found in correlation

Triton x 100, by Merck Group (13 mentions) Fv1000, by Olympus (5 mentions) Prolong gold antifade reagent, by Thermo Fisher Scientific (5 mentions) Paraformaldehyde (pfa), by Thermo Fisher Scientific (5 mentions) Vectashield hardset mounting medium, by Vector Laboratories (4 mentions) Lsm 710 confocal microscope, by Zeiss (4 mentions) Bovine serum albumin (bsa), by Merck Group (4 mentions) Dapi dye, by Vector Laboratories (3 mentions) Anti mouse lc3 a b, by Cell Signaling Technology (3 mentions) Paraformaldehyde (pfa), by Polysciences (3 mentions) Prolong gold antifade reagent, by Cell Signaling Technology (3 mentions) 4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (3 mentions) Prolong gold antifade mountant with dapi, by Thermo Fisher Scientific (3 mentions) Vectashield vibrance antifade mounting medium with dapi, by Vector Laboratories (2 mentions) Picro sirius red stain kit, by Abcam (2 mentions) Citrate buffer ph 6, by Vector Laboratories (2 mentions) Alexa fluor 488 donkey anti mouse, by Thermo Fisher Scientific (2 mentions) Masson s trichrome stain kit, by Polysciences (2 mentions) Donkey anti rat alexa fluor 647, by Thermo Fisher Scientific (2 mentions) Histo clear 2, by National Diagnostics (2 mentions)

Spelling variants of this product

Goat serum (26 citations) Normal goat serum (NGS; (3 citations) Normal serum (2 citations)

70 protocols using normal goat serum (ngs)

Quantifying Autophagy Markers in Cells

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Cells were plated at low confluence in 6 well plates (50,000 cells/well). On day 2, cells were exposed to serum starvation (0% FBS), normal medium (10% FBS), or chloroquine (50 μM) for 24 hours. Medium was removed, cells were washed with PBS and treated with 4% paraformaldehyde/PBS for 20 minutes at room temperature, washed, then permeabilized with 0.1% Triton X-100 for 10 minutes. Cells were then blocked with 5% normal goat serum (Cell Signaling Technology) containing 0.3% Triton X-100 in PBS for 60 minutes. Diluted primary antibody, anti-mouse LC3 A/B (Cell Signaling Technology), was applied in blocking buffer overnight at 4° C. Alexa Fluor-555 secondary antibody diluted in 1% normal goat serum in PBS were added for 1 hour at ambient temperature. Cells were fixed using Vectashield hard set mounting medium containing DAPI dye (Vector Laboratories). Images were acquired using confocal microscopy (Olympus FV-1000) and overlaid using ImageJ (22 (link)).

Clark C.A., Gupta H.B., Sareddy G., Pandeswara S., Lao S., Yuan B., Drerup J.M., Padron A., Conejo-Garcia J., Murthy K., Liu Y., Turk M.J., Thedieck K., Hurez V., Li R., Vadlamudi R, & Curiel T.J. (2016). Tumor-intrinsic PD-L1 signals regulate cell growth, pathogenesis and autophagy in ovarian cancer and melanoma. Cancer research, 76(23), 6964-6974.

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Klotho-α Immunohistochemistry in Brain

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PBS was perfused through the ascending aorta with PBS, followed by fresh 4% paraformaldehyde in PBS (pH 7.4) (n = 4). The brains were removed quickly and put in 4% paraformaldehyde in PBS for 24 h. After embedding the tissues with paraffin, they were sectioned at a thickness of 5 μm. Then, incubation was done with normal goat serum (1:100, Cell Signaling Technology Co. New York, USA); next, Klotho-α-specific antibody (1:100 dilution) covered the sections overnight at 4 °C followed by fluorescent secondary antibody Alexa Fluor 488-conjugated anti-rabbit IgG (1:500, Molecular Probes, Invitrogen, CA). A fluorescence microscope (Olympus IX71, Japan) was used for capturing the figures.

Shafie A., Rahimi A.M., Ahmadi I., Nabavizadeh F., Ranjbaran M, & Ashabi G. (2020). High-protein and low-calorie diets improved the anti-aging Klotho protein in the rats’ brain: the toxic role of high-fat diet. Nutrition & Metabolism, 17, 86.

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Autophagy Visualization Assay

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Cells were plated at low confluence in six‐well plates (50,000 cells/well). The following day, cells were exposed to serum starvation (EBSS), normal medium (10% FBS), or chloroquine (20 µM) for 6 h. Medium was removed, cells were washed with PBS and treated with 4% paraformaldehyde/PBS for 20 min at room temperature, washed, then permeabilized with 0.1% Triton X‐100 for 10 min. Cells were then blocked with 5% normal goat serum (Cell Signaling Technology) containing 0.3% Triton X‐100 in PBS for 1 h. Diluted (1:200) anti‐mouse microtubule‐associated protein 1A/1B‐light chain 3 (LC3) A/B (Cell Signaling Technology) was applied in blocking buffer overnight at 4°C. Alexa Fluor‐555 secondary antibody diluted in 1% normal goat serum in PBS was added for 1 h at ambient temperature. Cells were fixed using Vectashield hard set mounting medium containing DAPI (4’,6 ‐diamidino‐2‐phenylindole, Vector Laboratories). Images were acquired using confocal microscopy (Olympus FV‐1000) and overlaid using ImageJ.¹⁸

Zhang D., Reyes R.M., Osta E., Kari S., Gupta H.B., Padron A.S., Kornepati A.V., Kancharla A., Sun X., Deng Y., Wu B., Vadlamudi R., Li R., Svatek R.S, & Curiel T.J. (2021). Bladder cancer cell‐intrinsic PD‐L1 signals promote mTOR and autophagy activation that can be inhibited to improve cytotoxic chemotherapy. Cancer Medicine, 10(6), 2137-2152.

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Quantifying ZIKV Infection in Cells

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Productive ZIKV infection was qualitatively analyzed by IFA. Cells (2 × 10⁵) were infected at MOI 1, 10, or 20 PFU/cell with ZIKV and cultured on Lab-Tek II 4-well chamber slides (Fisher Scientific) at indicated time points. Cells were washed with 1× PBS and fixed with 4% paraformaldehyde for 15 min, then permeabilized with permeabilization buffer (BD Biosciences) for 30 min at room temperature. Cells were blocked with 10% normal goat serum (Cell Signaling Technology) for 1 h and then incubated with flavivirus envelope protein monoclonal antibody 4G2 (1:500; Millipore, MAB10216) for 2 h at room temperature. After incubation, cells were washed three times with PBS and incubated with Alexa Fluor 488-labeled anti-mouse antibody (Thermo Fisher, A11029) for 1 h at room temperature. Cells were then washed and mounted with Fluoroshield mounting medium containing 4′,6-diamidino-2-phenylindole (DAPI; Abcam, ab104139) for nuclei staining. All images were acquired by a fluorescence microscope (Olympus IX83).

Zwernik S.D., Adams B.H., Raymond D.A., Warner C.M., Kassam A.B., Rovin R.A, & Akhtar P. (2021). AXL receptor is required for Zika virus strain MR-766 infection in human glioblastoma cell lines. Molecular Therapy Oncolytics, 23, 447-457.

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Tissue Histology and Immunofluorescence Staining

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Tissues were fixed overnight in 10% formalin, dehydrated in ethanol, embedded in paraffin, and cut into 5 µm sections. Picrosirius Red staining was performed with the Picro Sirius Red Stain Kit (Abcam) according to the manufacturer’s instructions. Masson’s trichrome staining was performed with the Masson’s Trichrome Stain Kit (Polysciences) according to the manufacturer’s instructions. For immunofluorescence staining, sections were de-paraffinized with Histo-Clear II (National Diagnostics) and rehydrated according to the manufacturer’s instructions. Antigen retrieval was performed for 40 min in citrate buffer pH 6.0 (Vector Laboratories) in a steamer (IHC World). Sections were blocked in 5% BSA and 5% normal goat serum (Cell Signaling) in TBS containing 0.1% Tween-20, and incubated in primary antibodies at 4°C in a humidified chamber overnight. Sections were incubated in secondary antibody in blocking solution for 1h at room temperature and mounted in Vectashield Vibrance Antifade Mounting Medium with DAPI (Vector Laboratories). The following primary antibodies were used: SMA (1:400; Millipore, CBL171), CK8 (1:200; DSHB, TROMA-I). The following secondary antibodies were used: donkey anti-mouse Alexa-Fluor 488, donkey anti-rat Alexa Fluor 647 (1:1,000; Thermo Scientific).

Schwörer S., Pavlova N.N., Cimino F.V., King B., Cai X., Sizemore G.M, & Thompson C.B. (2021). Fibroblast pyruvate carboxylase is required for collagen production in the tumor microenvironment. Nature metabolism, 3(11), 1484-1499.

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Immunostaining of Mouse Cochlear Tissues

Cited 1 time

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Immunostaining was performed as previously described with minor modifications.^{7 (link),9} Briefly, tissue was fixed by transcardial perfusion of 4% paraformaldehyde (PFA, Alfa Aesar, MA), followed by harvest of the temporal bones and further fixation in 4% PFA at 4°C overnight. After adequate decalcification by incubation in 500mM EDTA (3–6 days), the cochlear ducts were dissected as described by the Eaton-Peabody Laboratories.¹² The tissue was permeabilized with PBS-0.3% Triton X-100 (Sigma-Aldrich, MO) and blocked in permeabilization buffer supplemented with 5% normal goat serum (Cell Signaling Technologies, MA). Pre-synaptic ribbons and post-synaptic densities were labelled using a monoclonal mouse anti-CtBP2 antibody (1:200, BD Biosciences, CA) and a monoclonal mouse anti-GluR2 antibody (1:2000, Sigma-Aldrich, MO), respectively. Following this, the tissue was incubated with the corresponding secondary antibodies, goat anti-mouse IgG2 Alexa Fluor^® 488 and goat anti-mouse IgG1 Alexa Fluor^® 568 (1:1000, ThermoFisher Scientific, MA). Nuclei were counterstained with DAPI. The labelled tissue was mounted with the ProLong Gold antifade reagent (ThermoFisher Scientific, MA).

Kennedy C.L., Shuster B., Amanipour R., Milon B., Patel P., Elkon R, & Hertzano R. (2023). Metformin Protects Against Noise-Induced Hearing Loss in Male Mice. Otology & Neurotology, 44(9), 956-963.

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Immunofluorescence Analysis of Endocrine Cells

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1.5×105 EndoC-βH1 cells were seeded on Matrigel and fibronectin coated 12-mm glass coverslips in 24-well plates and allowed to attach for 5 days at 37 °C. Cells were then vehicle treated or treated with the indicated drugs at the indicated experimental settings and fixed in 4% PFA (Polysciences, Inc.) in PBS for 15 minutes at room temperature. After being permeabilized by 0.5% Triton X-100 (Bio-Rad) in PBS for 10 minutes, cells were blocked for 1 hour with 1% normal goat serum (Cell Signaling) in PBS (both steps were done at room temperature). Cells were subsequently immunostained overnight at 4°C with the primary antibodies diluted in blocking buffer. Indirect immunofluorescence was performed using guinea pig polyclonal anti insulin, rabbit polyclonal anti C-peptide antibody, and rabbit monoclonal anti PDX-1 as described in Supplementary Table 2. Secondary antibodies conjugated to Alexa Fluor 488 or Alexa Fluor 568 (Life Technologies, 1:500) were used for 1 hour at room temperature. After final washes with PBS, the coverslips were mounted on microscope slides with ProLong Gold Antifade Reagent containing DAPI to counterstain the nuclei (Cell Signaling). Samples were examined with a Leica DM6000 microscope.

Lombardi A, & Tomer Y. (2017). Interferon alpha impairs insulin production in human beta cells via endoplasmic reticulum stress. Journal of autoimmunity, 80, 48-55.

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Immunohistochemical Analysis of Tumor Markers

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Tumors were harvested from MMTV-PyMT transgenic mice at the end point of treatment. Paraffinized tumor sections were permeabilized in 0.2% Triton X-100 after de-paraffinized in xylene and rehydrated in ethanol. Tumor sections were immersed in sodium-citrate buffer (10 mM, pH 6.0) for antigen retrieval and blocked in 5% normal goat serum (Cell Signaling Technology, Danvers, MA). The slides were then incubated with diluted primary antibodies Bax, MMP-7, PTEN, and β-catenin (Cell Signaling Technology, Danvers, MA) at 4 °C overnight. After washing, the slides were incubated with SignalStain Boost Detection Reagent (Cell Signaling Technology, Danvers, MA) and developed color according to SignalStain DAB Substrate Kit (Cell Signaling Technology, Danvers, MA) manufacturer’s guidelines. The study was conducted in accordance with the guidelines approved by Department of Health (Hong Kong, Ethic approval, 15–322). The images were detected by Leica DME microscope (Meyer Instruments, Inc., Houston, USA) and analyzed by ScopeImage 9.0 Image-Processing Software (BP Integrated Technologies, Inc., Calamba, PH).

Peng F., Tang H., Liu P., Shen J., Guan X., Xie X., Gao J., Xiong L., Jia L., Chen J, & Peng C. (2017). Isoliquiritigenin modulates miR-374a/PTEN/Akt axis to suppress breast cancer tumorigenesis and metastasis. Scientific Reports, 7, 9022.

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THIO Treatment Impacts M059K Cell Proliferation

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M059K cells were plated into a 24-well plate. 24 hours later, cells were treated with THIO at different concentrations, including 0, 1, 2.5, 5 μM. 96 hours after treatment, cells were fixed with 4% paraformaldehyde (ThermoFisher, 28908) for 15 minutes at room temperature. Cells were rinsed three times with 1× PBS for 5 minutes each, permeabilized with PBS containing 0.3% Triton X-100 (ThermoFisher, 9002-93-1) for 10 minutes and blocked with 5% normal goat serum (Cell Signaling Technologies, 5425) for 1 hour. Then, cells were incubated with anti-Ki-67 fluorescent antibody (Cell Signaling Technologies, 11882) overnight at 4 °C in the dark. Cells were rinsed three times with 1× PBS for 5 minutes each, stained with DAPI (Cell Signaling Technologies, 8961) for 5 minutes and then washed four times with 1× PBS. Images were obtained by a fluorescence microscope.

Yu S., Wei S., Savani M., Lin X., Du K., Mender I., Siteni S., Vasilopoulos T., Reitman Z.J., Ku Y., Wu D., Liu H., Tian M., Chen Y., Labrie M., Charbonneau C.M., Sugarman E., Bowie M., Hariharan S., Waitkus M., Jiang W., McLendon R.E., Pan E., Khasraw M., Walsh K.M., Lu Y., Herlyn M., Mills G., Herbig U., Wei Z., Keir S.T., Flaherty K., Liu L., Wu K., Shay J.W., Abdullah K., Zhang G, & Ashley D.M. (2021). A Modified Nucleoside 6-thio-2’-deoxyguanosine Exhibits Anti-tumor Activity in Gliomas. Clinical cancer research : an official journal of the American Association for Cancer Research, 27(24), 6800-6814.

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Quantifying Mcl-1 Expression in Senescent Cells

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Senescent melanocytes were treated with either DMSO or ABT-263 for 24 h. Then, cells were washed twice with PBS and fixed with 4% PFA for 10 min. Following two washes with PBS, cells were blocked in 5% normal goat serum (Sanquin) supplemented with 0.3% Triton X-100 for 1 h at room temperature and then they were incubated with primary antibody against Mcl-1 (1:800, Cell Signalling Technology, Ab#94296) diluted in 1% normal goat serum supplemented with 0.3% Triton X-100 overnight at 4 °C. The next day, cells were washed twice with PBS and incubated with goat anti-rabbit Alexa Fluor 488 (ThermoFisher Scientific, R37116) for 45 min at room temperature. Cells were washed in PBS and dH₂O before incubating in 2 μg/ml DAPI for 5 min. Cells were washed again in PBS before mounting with mounting medium. MCL-1 fluorescence was analyzed in >50 cells per condition and expressed as Corrected Total Cell Flourescence (CTCF). Using ImageJ, images were transformed to 8-bit, and cells were manually selected with a free drawing selection tool. The area, integrated density and mean gray value were measured in both the cells and in the background in each image. CTCF was calculated as Integrated Density – (Area of each selected cell · Mean fluorescence of the background reads).

Kohli J., Ge C., Fitsiou E., Doepner M., Brandenburg S.M., Faller W.J., Ridky T.W, & Demaria M. (2022). Targeting anti-apoptotic pathways eliminates senescent melanocytes and leads to nevi regression. Nature Communications, 13, 7923.

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