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Goat anti rat alexa fluor 647

Manufactured by Thermo Fisher Scientific
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Goat anti-rat Alexa Fluor 647 is a secondary antibody conjugated with the Alexa Fluor 647 fluorescent dye. It is designed to detect and bind to primary antibodies raised against rat antigens. The Alexa Fluor 647 dye provides a far-red fluorescent signal that can be detected using appropriate instrumentation.

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25 protocols using goat anti rat alexa fluor 647

1

Immunofluorescence Staining of Tight Junction Proteins

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BREC monolayers were fixed with 1% paraformaldehyde, permeabilized with 0.2% Triton X-100 and blocked with 10% goat serum, followed by their incubation with primary antibodies: polyclonal rabbit α-Claudin-5 (Invitrogen; 1:100), monoclonal mouse α-Occludin (Invitrogen; 1:100), monoclonal rat α-ZO-1 (Millipore; 1:100), or monoclonal mouse α-VE-Cadherin (F-8) (Santa Cruz Biotechnology; 1:100), for 2 days at 4°C. The primary antibodies were detected using secondary fluorescent antibodies: goat anti-mouse Alexa Fluor 488 (Life Technoligies, 1:400), goat anti-rabbit Alexa Fluor 594 (Life Technologies, 1:400), goat anti-rat Alexa Fluor 647 (Life Technologies, 1:400) and Hoechst (Life Technologies, 1:1000) overnight at 4 °C. Samples were imaged using a confocal microscope (TCS SP5; Leica, Wetzlar, Germany).
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2

Antibody Validation for Cell Signaling

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The following antibodies were used in this study: mouse anti-β-actin (Abcam, Cambridge, UK; cat# ab6276), mouse anti-γH2AX-S139 (Millipore, Burlington, MA, USA; cat# 05-636), mouse anti-c-Myc (Santa Cruz, Dallas, TX, USA; cat# sc-42), rabbit anti-cleaved PARP Asp214 (Cell Signaling, Danvers, MA, USA; cat# 9541), rabbit anti-MTH1 (Novus Biologicals, Centennial, CO, USA; cat# NB100-109), rabbit anti-p53 pS15 (Cell Signaling; cat# 9284), mouse anti-p53 (Santa Cruz; cat# sc-126), mouse anti-GAPDH (Abcam; cat# ab8245), rat anti-RPA32 (Cell Signaling; cat# 2208).
The secondary antibodies used were: goat anti-rat Alexa Fluor® 568 (Life Technologies, Carlsbad, CA, USA; cat# A-11077), goat anti-rat Alexa Fluor® 647 (Life Technologies; cat# A-21247), IRDye® 800CW donkey anti-rabbit (LI-COR, Lincoln, NE, USA; cat# 926-32213), IRDye® 680RD donkey anti-rabbit (LI-COR; cat# 926-68073), IRDye® 800CW donkey anti-mouse (LI-COR; cat# 926-32212), IRDye® 680RD donkey anti-mouse (LI-COR; cat# 926-68072).
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3

Immunostaining Fly Brain Neurons

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Fly brains were dissected in cold 0.3% PBST and fixed in 4% PFA in 0.3% PBST for 25 min at room temperature. Subsequently, brains were washed four to five times in 0.3% PBST and blocked in 10% normal goat serum (NGS) in 0.3% PBST for 1 hr at room temperature. Primary antibodies used were mouse anti-Bruchpilot Brp (nc82, Developmental Studies Hybridoma Bank, 1:20, RRID:AB_2314867), rabbit anti-dsRed (Takara Bio, 1:300, RRID:AB_10013483), and rat anti-Dα7 (gift from H. Bellen, 1:2000). Secondary antibodies used were: goat anti-mouse ATTO 647N (Rockland, 1:300, RRID:AB_2614870), goat anti-rabbit Alexa Fluor 568 (Thermo Fisher Scientific, 1:300, RRID:AB_10563601), and goat anti-rat Alexa Fluor 647 (Thermo Fisher Scientific, 1:300, RRID:AB_141778). GFP-labeled receptors were imaged natively without antibody staining. 5% NGS was added to all antibody solutions and both primary and secondary antibodies were incubated for at least 48 hr at 4°C. Brains were mounted in Vectashield Antifade Mounting Medium (Vector Laboratories) and imaged on a Leica TCS SP8 confocal microscope equipped with 488-, 561-, and 633 nm lasers, using a 63X glycerol objective.
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4

Immunostaining and Confocal Imaging of A549 Cells

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For immunostaining and imaging, A549 cells were cultivated on coverslips. The cells were fixed with 4% paraformaldehyde (PFA) for 20 min, permeabilized by 0.02% PBST (PBS with Triton X-100) for 20 min, washed 3X with PBS and blocked with 3% BSA in PBS for 30 min at room temperature. Anti-δ-hENaC antibody (1:500) was applied for 1 h at RT. After washing, cells were incubated with secondary antibody (goat anti-rat Alexa Fluor 647, Thermo Fisher Scientific), Vienna, Austria, for 1 h at RT in dark. Confocal images were acquired with a Zeiss LSM-510 confocal laser scanning microscope. Quantification was performed by ImageJ U.S. National Institutes of Health, Bethesda, Maryland, USA.
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5

Immunofluorescence and Western Blot Antibody Protocol

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In immunofluorescence experiments, the following antibodies and dilutions were used: anti-Phospho SQSTM1/p62 (S349) (Abcam, Cambridge, UK, cat # ab211324) 1:100; anti-SQSTM1/p62 (Abcam, cat # ab56416) 1:50; anti-β-Tubulin III (Merck Millipore, Burlington, MA, USA cat # T2200) 1:500; anti-MAP2 (1:500, Merck Millipore, cat # M9942) 1:500; anti-GFAP (Thermo Fisher Scientific, Waltham, MA, USA, cat # 13-0300), 1:800, donkey anti-rabbit-IgG Alexa Fluor 488 (Thermo Fisher Scientific, cat # R37118) 1:1000; donkey anti-mouse-IgG Alexa Fluor 594 (Thermo Fisher Scientific, cat # A-21203, 1:1000); goat anti-mouse IgG1 CF 568 (Merck, cat # SAB4600313 1:1000); goat anti-rat Alexa Fluor 647 (Thermo Fisher Scientific, cat # A21247, 1:1000); and DAPI (4′,6-diamidino-2-phenylindole Merck Millipore, cat #D9542) 1:5000. In Western blotting experiments, the following antibodies and dilutions were used: anti-Phospho SQSTM1/p62 (S349) (Abcam cat # ab211324) 1:2000; anti-SQSTM1/p62 (Abcam cat # ab56416) 1:2000; Anti-GAPDH (Abcam cat # ab8245); 1:5000; anti-phospho-Akt (Ser473 D9E Cell Signaling, Danvers, MA, USA, cat #4060) 1:2000; anti-Akt (Cell Signaling, cat # 9272, 1:1000); goat anti-mouse IgG IRDye 800(Li-Cor, Lincoln, NE, USA, cat # 926-32210) 1:5000; and goat anti-rabbit-IgG Alexa 680 (Thermo Fisher Scientific cat # A21076) 1:5000.
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6

Immunofluorescence Analysis of Lung Epithelial Cells

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CD49f+CD104+CD45-CD31-Ter119-GFP- cells were sorted from lung suspensions, cytospun on glass slides and fixed in 4% PFA in PBS for 10 min. Next, cells were permeabilized with 0.5% TritonX-100 for 30 min and incubated in blocking solution (4% BSA, 0.05% Tween20 in PBS) for 45 min at room temperature. Then, cells were incubated with a rat anti-E-cadherin antibody in blocking solution overnight at 4ºC followed by an incubation with a goat anti-rat AlexaFluor 647 (1:500, ThermoFisher Scientific (USA)). Finally, cells were mounted with Vectashield Mounting Medium with DAPI for imaging.
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7

Immunostaining of Neuronal Markers

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Adherent neurons were fixed in 4% paraformaldehyde for 5 min, followed by treatment with 0.5% saponin in PBS for 20 min for permeabilization. To block the samples, we treated the plates with 10% normal goat serum with 0.01% tween-20 in PBS for 30 min. Primary antibodies were then left incubating with the samples at 4°C overnight with 1% normal goat serum and 0.01% tween-20, before washing with PBS for three times. Secondary antibodies were then applied to 1% normal goat serum and 0.01% tween-20 at room temperature for 1 h before washing for another four times. The primary antibodies we used were Guinea pig anti-Synapsin I/II (Synaptic Systems, 1:500), rabbit anti-HOMER1 (Synaptic Systems, 1:500), chicken anti-MAP2 (Abcam, 1:1000), mouse anti-human nuclear antigen (Abcam, 1:200), rabbit anti-CUX2 (Abcam, 1:200) and rat anti-CTIP2 (Abcam, 1:500). The secondary antibodies we used were Goat anti-guinea pig Dylight 488 (Abcam), goat anti-mouse Alexa Fluor 488, goat anti-rabbit Alexa Fluor 555, goat anti-chicken Alexa Fluor 555, goat anti-rabbit Alexa Fluor 647 and goat anti-rat Alexa Fluor 647 (Thermo) at 1:1000 dilution.
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8

Whole-Mount Confocal Microscopy of Mice and Live-Imaging of Zebrafish

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For mice, whole-mount confocal microscopy was performed as described (Yokomizo et al. 2012 (link)). Embryos were stained with rat anti-CD31 (MEC13.3, BD Biosciences), rabbit anti-Runx (EPR3099, Abcam), chicken anti-GFP (Invitrogen Molecular Probes), and rat anti-F4/80 (CI: A3-1, Abcam) primary antibodies and goat anti-rat Alexa Fluor 555 (Abcam), goat anti-rabbit Alexa Fluor 488, goat anti-chicken Alexa Fluor 647 (Jackson Immunoresearch), and goat anti-rat Alexa Fluor 647 (Invitrogen Molecular Probes) secondary antibodies. Specimens were analyzed using a Zeiss LSM 710 confocal microscope with 25× objectives using multitrack sequential mode. The pinhole was set at 1 airy unit; for three-dimensional (3D) reconstructions, steps were 5 µm per z-section.
For zebrafish, fluorescent reporter embryos were treated as indicated in the text and live-imaged under tricaine anesthesia as previously described (North et al. 2007 (link)) using a Zeiss SteREO Discovery V8 microscope.
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9

Immunofluorescence Staining of Cells and Tissues

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For immunofluorescence staining, cells or tumor tissues from each treatment group were washed with ice-cold PBS and fixed with 4% paraformaldehyde (Electron Microscopy Sciences) in PBS for 20 min at room temperature, followed by permeabilization in 0.2% Triton X-100-PBS for 10 min. Samples were followed by blocking with PBS blocking buffer containing 2% normal goat serum, 2% BSA, and 0.2% gelatin for 1 h at room temperature. Then, the samples were incubated in primary antibodies at the appropriate concentration for 1 h at room temperature, washed with PBS and incubated in goat anti-rat-Alexa Fluor 647 (Molecular Probes) at 1:1000 dilution in blocking buffer for another 1 h at room temperature. Finally, stained cells were washed with PBS, counterstained with Hoechst 33342 (Molecular Probes-Invitrogen, H1399, 1:10000 dilution in PBS), and mounted on slides with Prolong Gold antifade mounting medium (Life Technologies). The slides were imaged under a confocal laser scanning microscope (Olympus, FV1100).
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10

Immunostaining Protocols for Ovary and Embryo

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The ovary and embryo immunostaining protocols used was previously described50 (link)51 (link). The primary antibodies used were as follows: mouse anti-GFP antibody (1:100; Roche), rabbit anti-Osk antibody (1:100; a gift from Dr. Tze-Bin Chou), rabbit anti-Tudor antibody (1:100; a gift from Dr. Akira Nakamura), rabbit anti-Krimp antibody (1:500; a gift from Dr. Toshie Kai) and rat anti-Vas antibody (1:100; Developmental Studies Hybridoma Bank). The fluorescently labelled secondary antibodies used were as follows: goat anti-mouse Alexa Fluor 488 (1:100; Invitrogen), goat anti-rabbit Alexa Fluor 647 (1:100; Invitrogen) and goat anti-rat Alexa Fluor 647 (1:100; Invitrogen).
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