A6455

Manufactured by Abcam

A6455 is a lab equipment product. It is a tool used for scientific research and analysis in a laboratory setting.

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Lab products found in correlation

A21271, by Merck Group (2 mentions) Click it alexa fluor 647 imaging kit, by Thermo Fisher Scientific (2 mentions) 4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions) Mab5258, by Merck Group (2 mentions) Sp5x scanning confocal microscope, by Leica (2 mentions) Ab4566, by BD (2 mentions) P40101, by Thermo Fisher Scientific (2 mentions) Fv1000 confocal microscope, by Olympus (1 mentions) Rabbit anti gfp, by Thermo Fisher Scientific (1 mentions) Chicken anti gfp, by Abcam (1 mentions) Ap09229pu n, by OriGene (1 mentions) Rabbit anti snai2, by Cell Signaling Technology (1 mentions) Gtx100118, by GeneTex (1 mentions) F3165, by GeneTex (1 mentions) S36973, by Thermo Fisher Scientific (1 mentions) A 11120, by Abcam (1 mentions) Goat anti rabbit hrp, by Bio-Rad (1 mentions) Chemidoc mp imaging system, by Bio-Rad (1 mentions) Rabbit anti gapdh, by Cell Signaling Technology (1 mentions) Ht7700, by Hitachi (1 mentions)

9 protocols using a6455

Assessing Cell Proliferation and Apoptosis in Zebrafish

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For EdU pulse labeling, Edu from the Click-iT Alexa Fluor 647 Imaging Kit (Life Technologies #C10340) was diluted to 2.5 mg/ml for retro-orbital injection. Each euthanized fish examined at 5 wpf was injected with 1 µl EdU solution, while 1.5 µl was used for 6 wpf fish. Fish were fixed 2 hours postinjection for cryo-sectioning; EdU detection was performed according to the manufacturer’s protocol.
Cryo-sectioning and immunofluorescence staining were performed as previously described.^{44 (link)} Antibodies against EGFP (Life Technologies #A6455 and #A11120), fibrillarin (Abcam #ab4566) and activated caspase-3 (BD Biosciences #559565) were used as primary antibodies. Secondary antibodies were conjugated with Alexa 488, 568, 647 (Life Technologies). DAPI (Life Technologies #S36973) was used for nuclear staining. Fluorescent images were taken by a Leica SP5X scanning confocal microscope at the Confocal and Light Microscopy core facility at Dana-Farber Cancer Institute.
Paraffin sectioning, H&E staining and immunohistochemistry with primary antibodies against TH (Pel-Freez #P40101), HuC/D (Life Technologies #A-21271) and synaptophysin (Millipore #MAB5258) were performed at the DF/HCC Research Pathology Core, and FACS at DFCI Flow Cytometry Core according to standard protocols.

Tao T., Sondalle S.B., Shi H., Zhu S., Perez-Atayde A.R., Peng J., Baserga S.J, & Look A.T. (2017). The pre-rRNA processing factor DEF is rate limiting for the pathogenesis of MYCN-driven neuroblastoma. Oncogene, 36(27), 3852-3867.

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Immunofluorescence Imaging of Drosophila Tissues

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Tissues were dissected in cold 1xPBS, fixed in 4% paraformaldehyde for 20-30 min at room temperature, and washed once quickly followed by three washes for 20 min each in PBS, 0.2% Triton-X100. Samples were blocked using 5% normal goat serum for 1–2 hr at room temperature and incubated with primary antibodies for a minimum of 24 hr at 4˚C, washed, and incubated with secondary antibody at 4˚C for 24 hr. Images were acquired using an Olympus FV1000 confocal microscope and analyzed by Volocity software to generate Z-stack projections and overlay images. The following antibodies were used for immunofluorescence studies: rat anti-dHNF4 (Palanker et al., 2009 (link)), rat anti-DILP2 (a gift from P. Leopold), rabbit anti-GFP (Molecular Probes A-6455), chicken anti-GFP (Abcam 13970), and mouse anti-ATP5A (Abcam 14748).

Barry W.E, & Thummel C.S. (2016). The Drosophila HNF4 nuclear receptor promotes glucose-stimulated insulin secretion and mitochondrial function in adults. eLife, 5, e11183.

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Immunostaining Embryos for Diverse Markers

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For immunostaining, embryos were fixed overnight at 4 °C with 4% formaldehyde (PFA) in phosphate-buffered saline (PBS) (pH 7.4), embedded in paraffin wax and serially sectioned at 8 μm. Immunostaining was performed either on whole mount embryos or on paraffin sections, as previously described (Burstyn-Cohen and Kalcheim, 2002 (link); Kahane and Kalcheim, 2020 (link)). Antibodies were diluted in PBS containing 5% fetal bovine serum (Biological Industries Israel, 04-007-1A) and 1% or 0.1% Triton X-100 (Sigma Aldrich Israel, X-100), respectively. Antibodies used were: rabbit anti-GFP (1:1000, Invitrogen, Thermo-Fisher Scientific, A-6455), mouse anti-GFP (1:100, Abcam, Ab38689), rabbit anti-RFP (1:1000, OriGene, AP09229PU-N), guinea pig anti-pSmad1/5/8 (1:300, a gift from E. Laufer), mouse anti-H3-pS10 (1:400, Abcam, Ab14955), rabbit anti-Laminin (1:100, Sigma Israel, L9393), rabbit anti-Sox9 (1:150, Millipore, AB5535), rabbit anti-SNAI2 (1:500; Cell Signaling Technologies, 9585), mouse anti-CD57 (anti-HNK1, 1:500, BD Biosciences, 559048), rabbit anti-BarHL1 (1:300, Sigma Israel, HPA004809), mouse anti-Pax7 (1:10, DHSB, pax7), mouse anti-Hb9 (1:200, DHSB, 81.5C10). Cell nuclei were visualized with 125 ng/ml Hoechst 33,258 (Sigma Israel, 14530) diluted in PBS.

Rekler D, & Kalcheim C. (2022). Completion of neural crest cell production and emigration is regulated by retinoic-acid-dependent inhibition of BMP signaling. eLife, 11, e72723.

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Antibody Immunofluorescence Staining

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Antibodies used for the study were as follows: anti-GFP (Molecular Probes, A6455), anti-Atg8 (Abcam, ab109364), anti-Flag (Sigma, F3165), and anti-GAPDH (GeneTex, GTX100118); antibody dilutions as indicated above.

Tang H.W., Spirohn K., Hu Y., Hao T., Kovács I.A., Gao Y., Binari R., Yang-Zhou D., Wan K.H., Bader J.S., Balcha D., Bian W., Booth B.W., Coté A.G., de Rouck S., Desbuleux A., Goh K.Y., Kim D.K., Knapp J.J., Lee W.X., Lemmens I., Li C., Li M., Li R., Lim H.J., Liu Y., Luck K., Markey D., Pollis C., Rangarajan S., Rodiger J., Schlabach S., Shen Y., Sheykhkarimli D., TeeKing B., Roth F.P., Tavernier J., Calderwood M.A., Hill D.E., Celniker S.E., Vidal M., Perrimon N, & Mohr S.E. (2023). Next-generation large-scale binary protein interaction network for Drosophila melanogaster. Nature Communications, 14, 2162.

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EdU Pulse Labeling and Tissue Analysis

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For EdU pulse labeling, EdU from the Click-iT Alexa Fluor 647 Imaging Kit (Life Technologies #C10340) was diluted to 2.5 mg/ml for retro-orbital injection. Each killed fish examined at 5 wpf was injected with 1 μl EdU solution, whereas 1.5 μl was used for 6 wpf fish. Fish were fixed 2 h post injection for cryo-sectioning; EdU detection was performed according to the manufacturer’s protocol.
Cryo-sectioning and immunofluorescence staining were performed as previously described.^{44 (link)} Antibodies against EGFP (Life Technologies #A6455 and #A11120), Fib (Abcam #ab4566) and activated caspase-3 (BD Biosciences #559565) were used as primary antibodies. Secondary antibodies were conjugated with Alexa 488, 568, 647 (Life Technologies). DAPI (Life Technologies #S36973) was used for nuclear staining. Fluorescent images were taken by a Leica SP5X scanning confocal microscope at the Confocal and Light Microscopy core facility at Dana-Farber Cancer Institute.
Paraffin sectioning, H&E staining and immunohistochemistry with primary antibodies against TH (Pel-Freez #P40101, Rogers, AR, USA), HuC/D (Life Technologies #A-21271) and synaptophysin (Millipore #MAB5258, Billerica, MA, USA) were performed at the DF/HCC Research Pathology Core, and fluorescence-activated cell sorting at DFCI Flow Cytometry Core according to standard protocols.

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Western Blot Analysis of GFP, WAC, and GAPDH

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HEK293T cells, cultured in DMEM with 10% FBS, were transfected with DNA plasmids using Lipofectamine^TM 2000. After 48 h, cells were collected and lysed in standard RIPA buffer with protease and phosphatase inhibitors and combined with Laemmli buffer (BioRad 1610737EDU) containing 2-mercaptoethanol and incubated at 95 °C for 5 min. Equal amounts of protein lysates were separated on 10% SDS-PAGE gels and then transferred to nitrocellulose membranes. The membranes were washed in Tris-buffered saline containing Triton X-100 (TBST) and blocked for 1 h in TBST containing 5% non-fat dry milk (blotto, sc-2324 SantaCruz biotechnology). Membranes were incubated with primary antibodies overnight at 4 °C, washed three times with TBST, incubated with secondary antibodies for 1 h at room temperature, and then washed three more times with TBST. Membranes were incubated in ECL solution (BioRad Clarity substrate 1705061) for 5 min, and chemiluminescent images were obtained with a BioRad Chemidoc™ MP imaging system. Antibodies (all used at 1:4000 dilution): rabbit anti-GFP (ThermoFisher A6455), rabbit anti-WAC (abCam ab109486), and rabbit anti-GAPDH (Cell Signaling Technology 2118), as well as goat anti-rabbit HRP (BioRad 170-6515).

Rudolph H.C., Stafford A.M., Hwang H.E., Kim C.H., Prokop J.W, & Vogt D. (2023). Structure-Function of the Human WAC Protein in GABAergic Neurons: Towards an Understanding of Autosomal Dominant DeSanto–Shinawi Syndrome. Biology, 12(4), 589.

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Immunostaining of Drosophila Larval Synapses

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The antibodies used in immunocytochemistry were anti-Bruchpilot (1:10, Developmental Studies Hybridoma Bank (DSHB), nc82, RRID:AB_2314867), anti-GluRIIA (1:10, DSHB, 8B4D2, RRID:AB_528269), anti-Dlg (1:250, DSHB, 4F3, RRID:AB_528203), anti-GFP (1:500, Thermo Fisher Scientific, A6455, RRID:AB_221570), anti-RFP (1:100, Abcam, ab62341, RRID:AB_945213), and Alexa Fluor594- (1:200) or DyLight649- (1:500) conjugated anti-horseradish peroxidase (HRP) (Jackson ImmunoResearch, 123-585-021, RRID:AB_2338966 and 123-495-021, discontinued). The visualizations of synapse boutons, mitoGFP and active zones (AZs) in larval motor neurons were analyzed by whole-mount immunostaining as previously described (Lee et al., 2010 (link)). For image processing of synapse boutons, thirty Z-stack images were taken at 0.15- to 0.30-μm intervals and were reconstituted using ImageJ. TEM images were obtained using an electron microscope (Hitachi, HT7700) at the Laboratory of Ultrastructural Research of Juntendo University.

Hosaka Y., Inoshita T., Shiba-Fukushima K., Cui C., Arano T., Imai Y, & Hattori N. (2017). Reduced TDP-43 Expression Improves Neuronal Activities in a Drosophila Model of Perry Syndrome. EBioMedicine, 21, 218-227.

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Immunofluorescence Staining of Cultured Cells

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Cells cultured in monolayers were fixed in 4% paraformaldehyde in PBS for 15 min at room temperature and iSKM bundles were fixed in 2% paraformaldehyde in PBS overnight at 4 °C while rocking. Following fixation, samples were washed twice with PBS, and kept in PBS at 4 °C for up to 1 week. Before staining, samples were blocked in PBS with 5% chick serum and 0.5% Triton-X 100. The following primary antibodies were used for immunostaining of: Oct4 (Millipore, MAB4401, 1:200), Tra-1-81 (Stemgent, 09-0011, 1:100), T (R&D, AF2085, 1:200), Pax3 (R&D, MAB2457, 1:200), Myf5 (SCBT, sc-302, 1:100), MF20 (DSHB, 1:300), sarcomeric α-actinin (SAA, Sigma, a7811, 1:200), GFP (Thermo, A6455, 1:300), laminin (Abcam, Cambridge, MA, ab11575, 1:200), Dystrophin (Abcam, Cambridge, MA, ab15277, 1:100), CD31 (Abcam, Cambridge, MA, ab28364, 1:50), MyoD (BD Biosciences, 554130, 1:100), MyoG (SCBT, sc-576, 1:100), and Pax7 (DSHB, 1:100). Corresponding fluorescently labeled secondary antibodies (1:500), α-bungarotoxin (B13422, 1:200), and phalloidin (O7466, 1:300) (all from Thermo) were applied in blocking solution (Supplementary Table 1) for 1 h. Images were acquired using a Leica SP5 inverted confocal microscope and analyzed using LSM Image Software.

Rao L., Qian Y., Khodabukus A., Ribar T, & Bursac N. (2018). Engineering human pluripotent stem cells into a functional skeletal muscle tissue. Nature Communications, 9, 126.

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Western Blot Analysis of Fluorescent Proteins

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Cells were pelleted by centrifugation at 120g for 6 min and resuspended in RIPA buffer for lysis and incubated at 4 °C for 10 min. Protein concentration was determined by BCA, matched across samples, and then run on SDS-PAGE followed by transfer using iBlot2. The membrane was blocked with 5% (w/v) skim milk in PBS-T for 1 h at room temperature followed by an incubation in either anti-GFP (Invitrogen # A-6455, 1:5000) or anti-Cherry antibody (Abcam # ab167453, 1:5000) in PBS-T for 1 h at room temperature. The blots were washed in PBS-T and then incubated with anti-rabbit secondary antibody (Invitrogen # 65–6120, 1:20,000) in PBS-T for 1 h at room temperature. Proteins were detected by an enhanced chemiluminescence kit (Clarity, Bio-Rad).

Kriachkov V., Ormsby A.R., Kusnadi E.P., McWilliam H.E., Mintern J.D., Amarasinghe S.L., Ritchie M.E., Furic L, & Hatters D.M. (2022). Arginine-rich C9ORF72 ALS proteins stall ribosomes in a manner distinct from a canonical ribosome-associated quality control substrate. The Journal of Biological Chemistry, 299(1), 102774.

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