A21209

Manufactured by Thermo Fisher Scientific

Sourced in United States

The A21209 is a laboratory instrument designed for the analysis and measurement of samples. It is a core piece of equipment used in various scientific workflows.

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Lab products found in correlation

29 protocols using a21209

Immunofluorescence Staining of Frozen Tissue

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The frozen sections were fixed with 4% paraformaldehyde (Sigma #P6148) and permeabilized with 0.25% Triton X-100 (Fisher Scientific #BP151–100). After a wash with PBS/Tween-20 (Fisher Scientific #BP337–100), slides were treated with 3 M hydrochloric acid (Fisher Scientific #A144S-500) for 10 min to open the nucleus structure if needed. Slides were blocked with 10% donkey serum (Sigma #D9663) in 2% BSA (Sigma) in PBS/Tw-20 for 1 h at room temperature. Afterwards, slides were probed with primary antibodies (CD31 1:25, BD #550274; BrdU-APC 1:50, BD; Alexa Fluor 488 Mouse anti-BrdU 1:10, BD #558599; Flag M2 1:500, Sigma #F1804) and incubated overnight at 4 °C. The next day, slides were washed and incubated with the fluorescence-conjugated secondary antibody (AF488 donkey anti-rat 1:300, Invitrogen #A-21208; AF594 donkey anti-mouse 1:300, Invitrogen #A-21203; AF594 donkey anti-rat 1:300, Invitrogen #A-21209). Images were taken with a confocal microscope LSM880 (Zeiss) and analyzed by Zen software (Zeiss).

Liu M., Zhang L., Marsboom G., Jambusaria A., Xiong S., Toth P.T., Benevolenskaya E.V., Rehman J, & Malik A.B. (2019). Sox17 is required for endothelial regeneration following inflammation-induced vascular injury. Nature Communications, 10, 2126.

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Immunostaining protocol for protein analysis

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For immunostaining, tissues were fixed in Zn-formalin for 24 hr and embedded in paraffin. Sections were deparaffinized, rehydrated, and prepared by antigen retrieval for 6 min each, and then blocked in 5% donkey serum for 1 hr at room temperature, incubated with either Rabbit anti-BNIP3 antibody at 1:100 (A5683, Abclonal), Rabbit anti-NF-kB-p65 1:400 (8242S, Cell Signaling Technology), Rabbit anti-Phospho-c-jun 1:200 (3270S, Cell Signaling Technology), Rat anti-ki67 1:100 (14-5698-82, ebiosciences), Rabbit anti-pRB-Ser807/811 1:400 (8516, Cell Signaling Technology), anti-p21 1:1000 (ZRB1141, Sigma), Rat anti-Phospho H3 1:1000 (H9908, Sigma), or Goat anti-GFP (ab6673, Abcam) at 1:250 overnight at 4°C, washed, incubated with secondary antibody AF594-anti-rabbit (A-21207, Invitrogen), Alexa594 anti-rat (A-21209, Invitrogen), and Alexa488 anti-goat (11055, Invitrogen) antibodies at 1:200 for 1 hr at room temperature, counterstained with DAPI, washed, and mounted. Slides were visualized and imaged using an Olympus IX71 inverted multicolor fluorescent microscope and a DP71 camera using ×400 magnification. MFI was calculated by measuring average intensity over a fixed threshold for all images. NF-kB staining was imaged via Zeiss LSM880 confocal microscope using ×63 oil-immersion objective, and intensity was measured on 0.8 µm z-sections.

Sela Y., Li J., Kuri P., Merrell A.J., Li N., Lengner C., Rompolas P, & Stanger B.Z. (2021). Dissecting phenotypic transitions in metastatic disease via photoconversion-based isolation. eLife, 10, e63270.

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Immunohistochemical Analysis of Tumor-Infiltrating Cells

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For CD3 and Gr1 staining, collected implanted tumor tissues were fixed in Zn-formalin for 24 hours and embedded in paraffin. Sections were deparaffinized, rehydrated, and prepared by antigen retrieval for 6 minutes each, and then blocked in 5% donkey serum (Sigma, D9663) for 1 hour at room temperature, incubated with primary antibodies overnight at 4°C, washed with 0.1% PBST (PBS with Tween-20), incubated with secondary antibodies for 1 hour at room temperature, and then washed and mounted. Slides were visualized and imaged using an Olympus IX71 inverted multicolor fluorescent microscope and a DP71 camera. For CD3 and Gr1 staining quantification, stained cells were counted for CD3+ T cells manually in 5–8 fields per sample.
Primary antibodies: CD3 (Abcam ab5690, 1:100 dilution) and Gr-1 (eBioscience 14-5931-85, 1:50 dilution), YFP (Abcam, ab6673). Secondary antibodies were purchased from Invitrogen (A-11055, A-21207, A-21209) and were used as 1:250 dilution for all staining.

Li J., Yuan S., Norgard R.J., Yan F., Sun Y.H., Kim I.K., Merrell A.J., Sela Y., Jiang Y., Bhanu N.V., Garcia B.A., Vonderheide R.H., Blanco A, & Stanger B.Z. (2020). Epigenetic and transcriptional control of the epidermal growth factor receptor (EGFR) regulates the tumor immune microenvironment in pancreatic cancer. Cancer discovery, 11(3), 736-753.

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Immunofluorescence Staining of BV-2 Cells

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The BV-2 cells were fixed in 4% paraformaldehyde for 30 min at room temperature and washed with PBS. Then the non-specific binding sites were blocked with 10% goat serum (GTX27481, Gene-Tex, USA) for 1 h at room temperature, and the samples were then incubated at 4 °C overnight with the following primary antibodies in PBS: iNOS (1:100, ab15323, Abcam, UK), Ym-1(1:100, ab192029, Abcam, UK), and F4/80 (1:50, ab6640, Abcam, UK). The following day, sections were incubated with mouse anti-rabbit IgG (1:1000, #4408/4409, Cell Signaling Technology, USA), rabbit anti-mouse IgG (1:1000, #4412/4413, Cell Signaling Technology, USA), and donkey anti-rat IgG (1:1000, A21209, Invitrogen, USA) for 1 h at room temperature. After rinsing, sections were mounted with a DAPI-containing antifade solution. Fluorescence signals were then observed under a microscope (BX63; Olympus) and a confocal microscope (Zeiss; LSM800).

Wang J., Pan H., Lin Z., Xiong C., Wei C., Li H., Tong F, & Dong X. (2020). Neuroprotective Effect of Fractalkine on Radiation-induced Brain Injury Through Promoting the M2 Polarization of Microglia. Molecular Neurobiology, 58(3), 1074-1087.

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Antibody Reagents for Protein Analysis

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The following antibodies and working concentration were used in this study: anti-CTTNBP2 (A5, A7, and 9W, rabbit, homemade, 0.5 μg/ml) [5 (link), 20 (link)], anti-Myc tag (9B11, Cell Signaling Technology, 1/1000 for staining; 06-549, Millipore, 1 μg/ml), anti-HA tag (3F10, Roche, 0.5 μg/ml), anti-HA tag (Y-11, Santa Cruz Biotechnology, 0.5 μg/ml), anti-GFP (ab13970, Abcam, 0.5 μg/ml), anti-αtubulin (B-5-1-2, Sigma-Aldrich, 1 μg/ml), anti-acetyl tubulin (6-11B-1, Sigma-Aldrich, 1 μg/ml), anti-βactin (AC-74, Sigma-Aldrich, 1/1000), anti-cortactin (H-191, Santa Cruz Biotechnology, 0.5 μg/ml), anti-FOS (#2250, clone 9F6, Cell Signaling Technology, 1/200), anti-mouse HRP (NA931, GE Healthcare, 1/5000), anti-rabbit HRP (NA934, GE Healthcare, 1/5000), anti-chicken Alexa Fluor 488 (A-11039, Invitrogen, 1 μg/ml), anti-mouse Alexa Fluor 555 (A-21424, Invitrogen, 1 μg/ml), anti-rat Alexa Fluor 594 (A-21209, Invitrogen, 1 μg/ml), and anti-rabbit Alexa Fluor 647 (A-21244, Invitrogen, 2 μg/ml).

Shih P.Y., Hsieh B.Y., Tsai C.Y., Lo C.A., Chen B.E, & Hsueh Y.P. (2020). Autism-linked mutations of CTTNBP2 reduce social interaction and impair dendritic spine formation via diverse mechanisms. Acta Neuropathologica Communications, 8, 185.

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Immunohistochemical Staining of Tissue Sections

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Tissue sections were permeabilized using 0.5% TritonX-100 in PBS for 20 min at room temperature. To prevent non-specific binding of antibodies, sections were blocked using 10% BSA in PBS for 1 h at room temperature. Primary antibodies were diluted in an antibody buffer solution (0.3% Tween-20 in PBS (PBST), 10% donkey serum), applied to the blocked sections, and allowed to incubate overnight at 4 °C. After three washes of 0.3% PBST, fluorescently labeled secondary antibodies diluted in the antibody buffer were added and incubated for 1 h at room temperature. Samples were washed three times with 0.3% PBST and once with PBS, before being mounted with Vectashield antifade mounting medium. Antibodies, along with their source and dilution, used in this study are as follows: CD31 (Abcam, ab182981, 1:500), CD68 (Invitrogen, FA-11, 1:250), CD206 (Proteintech, 18704–1-AP, 1:250), Laminin (Abcam, ab11576, 1:500), Neurofilament (Invitrogen, MA5–14981, 1:250), Alexa Fluor 594 Donkey anti-Rat (Invitrogen, A21209, 1:500), Alexa Fluor 647 Donkey anti-Rabbit (Invitrogen, A21207, 1:500).

Eugenis I., Wu D., Hu C., Chiang G., Huang N.F, & Rando T.A. (2022). Scalable macroporous hydrogels enhance stem cell treatment of volumetric muscle loss. Biomaterials, 290, 121818.

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Immunohistochemical Analysis of Tumor Markers

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For CD3, USP22, and Gr1 staining, collected implanted tumor tissues were fixed in Zn-formalin for 24 hours and embedded in paraffin. Sections were deparaffinized, rehydrated, and prepared by antigen retrieval for 6 minutes each, and then blocked in 5% donkey serum (Sigma, D9663) for 1 hour at room temperature, incubated with primary antibodies overnight at 4°C, washed with 0.1% PBST (PBS with Tween-20), incubated with secondary antibodies for 1 hour at room temperature, and then washed and mounted. Slides were visualized and imaged using an Olympus IX71 inverted multicolor fluorescent microscope and a DP71 camera. For CD3 and Gr1 staining quantification, stained cells were counted for CD3⁺ T cells manually in 5-8 fields per sample.
Primary antibodies: CD3 (Abcam ab5690, 1:100 dilution), USP22 (Abcam ab195289, 1:100 dilution), Gr-1 (eBioscience 14-5931-85, 1:50 dilution), YFP (Abcam, ab6673). Secondary antibodies were purchased from Invitrogen (A-11055, A-21207, A-21209) and were used as 1:250 dilution for all staining.

Li J., Yuan S., Norgard R.J., Yan F., Yamazoe T., Blanco A, & Stanger B.Z. (2019). Tumor cell–intrinsic USP22 suppresses antitumor immunity in pancreatic cancer. Cancer immunology research, 8(3), 282-291.

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Immunostaining of Embryonic Gonad Sections

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Embryonic gonads were fixed in 4% PFA for 30 min at 4°C. Gonads were next submerged in 10 and 20% sucrose in PBS for 1 h each at 4°C, and in 30% sucrose in PBS overnight at 4°C. The gonads were then embedded in Tissue-Tek O.C.T. compound (Sakura Finetek) and frozen in liquid nitrogen. Six-micrometer-thick sections of each gonad were applied to glass slides and autoclaved in TRS (Dako). After pre-incubation with 3% skim milk in PBS-T (PBS with 0.1% Tween20 (Sigma-Aldrich)) at RT for 30 min, the sections were reacted with primary antibodies overnight at 4°C at the following dilutions: 1:200 for mouse anti-phospho-histone H3 (ser10) (1:200, Sigma-Aldrich, 06-570), goat anti-CDH1 (1μg/ml, R&D, AF748), rabbit anti-DNMT3L (1:500, provided by Dr. Yamanaka), rabbit anti-STRA8 (1:200, abcam #ab49602), rabbit anti-Ki67 (1:200, Invitrogen #MA5-14520), rat anti-Ki67 (1:100, Invitrogen AB_10854564) and anti-pS6 (1:200, CST #2211). Secondary antibodies labeled with Alexa 488, 594 or 647 (1:1,000, Invitrogen A21207, A21208, A21209, A32766, A32814, A32754, A32744 and A32795) were used. DNA was counter-stained with DAPI (100 ng/ml). Fluorescence microscopy was performed using Olympus FV1200 and images were processed with ImageJ/Fiji (Schindelin et al., 2012 (link)).

Shimada R., Koike H., Hirano T., Kato Y, & Saga Y. (2021). NANOS2 suppresses the cell cycle by repressing mTORC1 activators in embryonic male germ cells. iScience, 24(8), 102890.

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Multiplex Immunofluorescence Staining Protocol

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For immunofluorescence double staining, quenching of endogenous peroxidase and blocking of endogenous biotin were omitted. Sections were incubated with anti-TTF1 SP141 (1:200; Abcam ab227652) and anti-P40(1:1000; a gift from Dr. Philip J Coates, Masaryk Memorial Cancer Institute, Czech Republic); or with anti-MMP12 (1:100; Novus Bio, NBP2-67344) and anti-F4/80 (1:100; Bio-Rad, MCA497GA); or with anti-IL-1β (1:100; abcam, ab283818) and anti-F4/80( 1:100; Bio-Rad, MCA497GA); or with anti-Arg1(1:100; Santa Cruz, sc-18351) and anti-F4/80 (1:1000; Bio-Rad, MCA497GA); or with anti-IL-1β (1:100; abcam, ab283818) and anti-Arg1(1:100; Santa Cruz, sc-18351). Primary antibodies were detected with donkey anti-rabbit FITC (1:400; Invitrogen, A21206) and donkey anti-mouse 594 (1:400; Invitrogen, A21203) or donkey anti rabbit FITC (1:400; Invitrogen, A21206) and donkey anti-rat 594 (1:400; Invitrogen, A21209); donkey anti rabbit FITC (1:400; Invitrogen, A21206) and donkey anti-goat Cy3 (1:400; Jackson ImmunoResearch, 05-165-147). Samples were covered with medium containing with 4', 6'-diamidino-2-phenylindole (DAPI) (Vector) for cell nuclei visualization. The Keyence microscope (BZ-X810) was used to take the images.

Wu W., Sarhadi M., Song X., Xue J., Dai Y, & Gustafsson J.A. (2023). Liver X receptors and estrogen receptor β, two players in a rare subtype of NSCLC. International Journal of Biological Sciences, 19(9), 2848-2859.

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Immunostaining of Mouse Lung Tissue

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Sections of mouse lung tissue 3 µm thick were prepared for immunofluorescence staining using primary antibodies against inducible nitric oxide synthase (iNOS; NOS2) (sc-7271; Santa Cruz Biotechnology), F4/80 (sc-52664; Santa Cruz Biotechnology), and appropriate secondary antibodies (A11029 and A21209; Invitrogen). Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (P36935; Invitrogen). Immunofluorescence images were acquired using a Leica TCS SP8 microscope system (Leica).

Kim Y.J., Lee J.Y., Lee J.J., Jeon S.M., Silwal P., Kim I.S., Kim H.J., Park C.R., Chung C., Han J.E., Choi J.W., Tak E.J., Yoo J.H., Jeong S.W., Kim D.Y., Ketphan W., Kim S.Y., Jhun B.W., Whang J., Kim J.M., Eoh H., Bae J.W, & Jo E.K. (2022). Arginine-mediated gut microbiome remodeling promotes host pulmonary immune defense against nontuberculous mycobacterial infection. Gut Microbes, 14(1), 2073132.

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