A11012

Manufactured by Thermo Fisher Scientific

Sourced in United States, United Kingdom, France

The A11012 is a laboratory equipment designed for performing essential functions in a scientific research environment. It serves as a reliable tool for users to conduct their experiments and analyses.

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Alexa Fluor™ 594 goat anti-rabbit IgG(H + L) A11012

135 protocols using a11012

Penile Tissue Histological Analysis

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Penile midshaft tissue was freshly harvested and divided into three parts transversally. One part was cryopreserved for protein extraction, one part was cryo-embedded for immunofluorescent (IHF) staining, and the final part was paraffin embedded for immunohistochemical (IHC) staining. For IHC and IHF, tissue sections used were 5 μm thick. Dilutions of primary antibodies (incubated overnight at 4°C) were as follows: alpha-smooth muscle actin (α-SMA; 1:3000; ab124964, Abcam, Cambridge, UK); neuronal-nitric oxide synthase (n-NOS; 1:500; ab95436, Abcam); neurofilament medium (NF; 1:500; ab7794, Abcam); and rat endothelial cell antigen-1 (Reca-1; 1:500; HIS52, Bio-Rad, Hercules, CA, USA). Appropriate species-directed secondary antibodies were applied to the sections for 90 min at room temperature; for IHF: α-SMA (1:500; A11012, Invitrogen, Carlsbad, CA, USA), n-NOS (1:500; A11001, Invitrogen); NF (1:500; A11012, Invitrogen), and Reca-1 (1:500; A11001, Invitrogen); and for IHC: α-SMA (ZB-2301, ZSGB-BIO, Beijing, China), nNOS (ZB-2305, ZSGB-BIO), NF (ZB-2301, ZSGB-BIO), and Reca-1 (ZB-2305, ZSGB-BIO). Semi-quantitative analysis was performed to evaluate the intensity of α-SMA, nNOS, NF, and Reca-1 staining using Image Pro Plus software (version 6.0, Media Cybernetics Corporation, Houston, TX, USA).

Li M., Yuan Y.M., Yang B.C., Gu S.J., Li H.X., Xin Z.C., Fang D, & Guan R.L. (2020). Comparative study of intracavernous pressure and cavernous pathology after bilateral cavernous nerve crushing and resection in rats. Asian Journal of Andrology, 22(6), 629-635.

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Immunofluorescence Staining of Spheroids

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Spheroids were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100 in PBS. Following fixation, cells were incubated with antibodies against Sox2 (#4900; Cell Signaling), E-cadherin (BD610181; BD Biosciences), p-MEK1/2 (#9121; Cell Signaling), Slug (#9585; Cell Signaling), and/ or CD44-FITC (555478; BD Biosciences), in a solution of PBS with 1% BSA and 0.1% Triton X-100 at 4 °C overnight. Staining was visualized using anti-mouse AlexaFluor 488 (A11005; Life Technologies) and anti-rabbit AlexaFluor 594 (A11012; Life Technologies) with nuclear counterstaining using DAPI, and imaging on an inverted confocal microscope. Images were processed using Imaris 7.6.

Yoon C., Till J., Cho S.J., Chang K.K., Lin J.X., Huang C.M., Ryeom S, & Yoon S.S. (2019). KRAS activation in gastric adenocarcinoma stimulates epithelial-to-mesenchymal transition to cancer stem-like cells and promotes metastasis. Molecular cancer research : MCR, 17(9), 1945-1957.

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Immunostaining of Piezo2 and GFP in Mouse Brain

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Mice were anesthetized with ketamine (200 mg/ml) and xylacine (50 mg/ml), and they were perfused with 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (PB). The brain was then removed from each mouse and after post-fixing by immersion in 4% PFA (24 h, 4 °C), coronal vibratome sections (50 μm; VT 1000S, Leica) were obtained and then incubated for 30 min in blocking solution at room temperature (3% goat serum in TTBS). The sections were immunostained overnight at 4 °C with the primary antibodies and after washing three times in PBS (5 min) the secondary antibodies were applied for 2 h at room temperature. After three 5 min rinses with PBS, the nuclei were stained with DAPI (2 μM; Life Technology, Carlsbad, USA). The antibodies used were: rabbit anti-Piezo2 (1:100; NBP1-78624, Novus Biological, Littleton, USA); chicken anti-GFP (1:1000; AB13970, Abcam, Cambridge, UK), goat-anti rabbit AlexaFluor-594 (1:1000; A11012) and goat-anti chicken AlexaFluor-488 (1:2000; A11039, Life Technology).

Florez-Paz D., Bali K.K., Kuner R, & Gomis A. (2016). A critical role for Piezo2 channels in the mechanotransduction of mouse proprioceptive neurons. Scientific Reports, 6, 25923.

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Immunofluorescence Staining of Neural Markers

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Immunofluorescence staining was performed as described [48 (link)]. Primary antibodies were as follows: anti-Nestin (839801, Biolegend; 1:1000 dilution) and anti-Tubb3 (801201, Biolegend; 1:1000 dilution). Secondary antibodies were as follows: goat anti-mouse Alexa Fluor 488 (A-11011, Life Technologies; 1:1000 dilution) and goat anti-rabbit Alexa Fluor 594 (A-11012, Life Technologies; 1:1000 dilution).

Llères D., Moindrot B., Pathak R., Piras V., Matelot M., Pignard B., Marchand A., Poncelet M., Perrin A., Tellier V., Feil R, & Noordermeer D. (2019). CTCF modulates allele-specific sub-TAD organization and imprinted gene activity at the mouse Dlk1-Dio3 and Igf2-H19 domains. Genome Biology, 20, 272.

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Immunofluorescence and Endocytic Trafficking Analysis

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For immunofluorescence analysis, the cells were fixed with PBS/PFA 4% for 10 min and permeabilized with PBS/Triton 100 × 0.2% for 5 min. After blocking with PBS/BSA 0.2% for 1 hr, the cells were then incubated with primary antibodies (1/100) at room temperature for 1 hr. Secondary antibodies coupled with Alexa-594 and/or Alexa-488 (A-11012, A-11001, Life technologies) were used at 1/500 for 1 hr. Nuclei were counterstained with DAPI (Sigma-Aldrich). For the pulse-chase experiments, the cells were incubated for 10 min with 100 ng/ml of EGF-alexa647 or 40 ng/ml of HGF-Alexa647 with at 4°C and chased with fresh media for 0, 5, 10, 30, 60, 120, 180, and 240 min at 37°C (Collinet et al., 2010 (link)). Then, the cells were washed, fixed, and immunolabeled as described above. Images were obtained in a randomized fashion using an LSM510 confocal microscope (Zeiss) and a Nikon A1R confocal. At least 20 images per condition were analysed using the unbiased multiparametrics MotionTracking software, as previously described (generous gift from Dr Y. Kalaidzidis, Marino Zerial’s lab) (Gilleron et al., 2013 (link); Zeigerer et al., 2012 (link)).

Torrino S., Tiroille V., Dolfi B., Dufies M., Hinault C., Bonesso L., Dagnino S., Uhler J., Irondelle M., Gay A.S., Fleuriot L., Debayle D., Lacas-Gervais S., Cormont M., Bertero T., Bost F., Gilleron J, & Clavel S. (2021). UBTD1 regulates ceramide balance and endolysosomal positioning to coordinate EGFR signaling. eLife, 10, e68348.

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Immunofluorescence Staining of Adherent Cells

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Cells were plated at 20,000 cells/well on coverslips in 24 well plates and allowed to attach overnight. Wells were fixed, permeabilized, and blocked as described previously [54 ]. Cells stained on cover slips were mounted on slides using Fluorescence Mounting Medium (S3023, DAKO). The coverslips were then incubated for 16 hours at 4°C with polyclonal rabbit anti-CK19 (10 μg/ml, ab15463, Abcam) or polyclonal rabbit anti-N-cadherin (1:100, ab76057, Abcam). Alexa Fluor 594 polyclonal goat anti-rabbit (1:700, A-11012, Life Technologies) was used as a secondary antibody in 1% BSA (A2153, Sigma-Aldrich) in PBS for 1 hour at room temperature. For L1CAM, monoclonal anti-L1CAM-PE (1:50, ab95694, Abcam) was used. Nuclei were counterstained with DAPI (1 μg/ml, 10236276001, Roche,), in PBS for 5 minutes at room temperature. Images were obtained using an Axiovert 200M fluorescence microscope (Zeiss Axiovert 200 M, Carl Zeiss MicroImaging GmbH, Jena, Germany) with CCD camera (Zeiss Axiocam HR), using Axiovision software. All images are taken with 40x magnification.

Lund K., Dembinski J.L., Solberg N., Urbanucci A., Mills I.G, & Krauss S. (2015). Slug-Dependent Upregulation of L1CAM Is Responsible for the Increased Invasion Potential of Pancreatic Cancer Cells following Long-Term 5-FU Treatment. PLoS ONE, 10(4), e0123684.

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Progerin and Lamin A/C Immunofluorescence

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Fibroblasts were seeded into 4 chamber-well slides (SPL Lifesciences, Korea). Cells were fixed for 15 minutes (RT) in a 4% paraformaldehyde +2% sucrose solution and then permeabilized for 3 minutes at RT using permeabilization buffer (0.5% Triton X-100, 50 mM NaCl, 300 mM sucrose, 20 mM HEPES pH 7.5, 3 mM MgCl2). Cells were then incubated with primary antibodies for 40 minutes at 37 °C (anti-progerin (1/50, sc-81611) and anti-lamin A/C (1/50, sc-20681), Santa Cruz Biotechnology). After washing, cells were incubated for 20 minutes at 37 °C with secondary antibodies (A11001, A11012, 1/400 Life Technologies). Nuclei were stained with DAPI for 10 minutes at room temperature (0.1 μg/mL, Thermofisher). Slides were mounted using FluorSave™ reagent (Merck Millipore) and observed on a fluorescence microscope (ApoTome.2 Zeiss).

Frankel D., Delecourt V., Novoa-del-Toro E.M., Robin J.D., Airault C., Bartoli C., Carabalona A., Perrin S., Mazaleyrat K., De Sandre-Giovannoli A., Magdinier F., Baudot A., Lévy N., Kaspi E, & Roll P. (2022). miR-376a-3p and miR-376b-3p overexpression in Hutchinson-Gilford progeria fibroblasts inhibits cell proliferation and induces premature senescence. iScience, 25(2), 103757.

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DNA Damage Response Imaging Protocol

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Cells were cultured on coverslips and were treated with 10 μM VP-16 for the indicated times. Cells were then washed with PBS, fixed with 4% paraformaldehyde for 10 min at room temperature, and were subsequently permeabilized in 0.5% Triton X-100 solution for 10 min. After being blocked by 5% BSA, cells were incubated with primary antibodies overnight and subsequently secondary antibodies for 1 h. Coverslips were then mounted using DAPI containing anti-fade. Images were captured using a Leica SP8 Laser confocal optical imaging platform. Images were processed using Leica Application Suite X. The percentage of cells carrying indicated foci was calculated after analyzing three independent experiments. Approximately 150 cells were counted for each sample. Antibodies used for immunofluorescent staining are as follows: anti-gamma-H2AX (Abcam; Mouse; ab22551; 1:100 dilution), anti-Rad51(Abcam; Rabbit; ab133534; 1:400 dilution), anti-Brca1(Santa Cruz Biotechnology; Mouse; sc-6954; 1:100 dilution), and anti-FLAG (Cell Signaling Technology; Rabbit; #14793; 1:200 dilution). The following secondary antibodies were used: Alexa Fluor 488 Goat Anti-Mouse (Life Technologies; A-11008; 1:1000) and Alexa Fluor 594 Goat Anti-Rabbit (Life Technologies; A-11012; 1:1000).

Liu G., Li J., He B., Yan J., Zhao J., Wang X., Zhao X., Xu J., Wu Y., Zhang S., Gan X., Zhou C., Li X., Zhang X, & Chen X. (2023). Bre1/RNF20 promotes Rad51-mediated strand exchange and antagonizes the Srs2/FBH1 helicases. Nature Communications, 14, 3024.

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Immunohistochemical Analysis of Mouse Brain Tissue

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Mice were anesthetized with isoflurane and fixed via transcardial perfusion with 4% paraformaldehyde (PFA) in phosphate buffered saline (PBS). After dissection, the brain tissue was fixed in 4% PFA for 24 hr at 4°C. After a wash in PBS, the fixed tissue was placed in a sucrose solution (30% w/v in PB) for 48 hr at 4°C until tissue sinks. This tissue was embedded in O.C.T. compound (Cat. Num. 4586, Scigen), frozen on dry ice and stored at −80°C until processing. The brain tissues were sliced into 30-μm thick coronal slices using a Leica CM 1850 cryostat (Leica, Wetzlar, Germany). These free-floating sections of the cortex, striatum and hippocampus were blocked with PBS containing 0.3% Triton X-100 and 10% SuperBlock (Cat. Num. 37515, Life Technologies) followed by incubation with primary antibodies for 24 to 48h at 4°C. After the sections were rinsed with a solution of 0.3% Triton in PBS, the slices were incubated with the secondary antibodies: goat anti-rabbit Alexa Fluor 594 (1:3000, A11012, Life Technologies) or donkey anti-rabbit Alexa Fluor 488 (1:1000, A21206, Life Technologies), donkey anti-mouse Alexa Fluor 594 (1:3000, A21203, Life Technologies), and goat-anti-chicken Alexa Fluor 488 (1:1000, A11039, Life Technologies). Hoechst as a nuclear counterstain was used.

Iemolo A., Montilla-Perez P., Lai I.C., Meng Y., Nolan S., Wen J., Rusu I., Dulcis D, & Telese F. (2020). A cell type-specific expression map of NCoR1 and SMRT transcriptional co-repressors in the mouse brain. The Journal of comparative neurology, 528(13), 2218-2238.

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Fluorescent Labeling of Hippocampal Neurons

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Cell filling was performed as described in previous methods59 (link)60 (link). Briefly 200 μm sections were cut from a 4% PFA perfused fixed brain using a vibratome and cells were injected with a continuous current of Alexa Fluor 594 dye solution (Life Technologies) in CA1, CA3 and DG region of the hippocampus. This fluorescent marker was applied, with the aid of an electrode, by continuous current until the distal tips of each dendrite fluoresced brightly, thereby ensuring that the dendrites were completely filled and the fluorescence did not diminish at a distance from the soma. For immunohistochemistry, brain sections were blocked and permeabilised in 5% bovine serum albumin (BSA, Sigma-Alrich) and 0.2% Triton X-100. Anti-synaptophysin I (1:250, ab14692, Abcam) used in solution of 3% BSA, 0.2% Triton X-100 at 4 °C overnight. Secondary antibody incubation of Alexa Fluor 594 (1:500, A-11012, Life Technologies) was performed for 1 hour at room temperature. Sections were then washed in 1x PBS and mounted.

Broadhead M.J., Horrocks M.H., Zhu F., Muresan L., Benavides-Piccione R., DeFelipe J., Fricker D., Kopanitsa M.V., Duncan R.R., Klenerman D., Komiyama N.H., Lee S.F, & Grant S.G. (2016). PSD95 nanoclusters are postsynaptic building blocks in hippocampus circuits. Scientific Reports, 6, 24626.

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