We used the following primary antibodies: rabbit polyclonal antibodies against SANS (RpAb) (29 (link),37 (link)), anti-SANS (mouse, monoclonal antibody; MmAb) (sc-514418, Santa Cruz), anti-FLAG (F1804, Sigma Aldrich), anti-PRPF6 (sc-48786, Santa Cruz), anti-HA (Roche), anti-GFP (ab6556, Abcam), anti-PRPF31 (PAB7154, Abnova), anti-hSNU114 (38 (link)), anti-Coilin (sc-55594, Santa Cruz), anti-SC35 (sc-53518, Santa Cruz), anti-SON (ATLAS, HPA023535), anti-SF3B1 (MmAb) (sc-514655, Santa Cruz), anti-SF3B1 (RmAb) (Will et al. 2001), anti-U5 52K (39 (link)), anti-SART1 (PA556663, Thermo Fisher) and anti-PRPF38 (40 (link)). Secondary antibodies were conjugated to the following fluorophores: Alexa488 (donkey-anti-rabbit, A21206, Molecular Probes,), CF 488 (donkey-anti-guinea pig, 20169-1, Biotrend,), Alexa488 (donkey-anti-mouse, A21202, Molecular Probes), Alexa 555 (donkey-anti-mouse, A31570, Molecular Probes), Alexa 568 (donkey-anti-rabbit, A10043, Invitrogen,) CF640 (donkey-anti-goat, 20179-1, Biotrend), Alexa 680 (donkey-anti-goat, A21084, Molecular Probes), Alexa 680 (goat-anti-rat, A21096, Molecular Probes) Alexa 680 (donkey-anti-rabbit, A10043), IRDye 800 (donkey-anti-mouse, 610–732-124, Rockland), Abberior STAR Orange (goat-anti-mouse, Abberior,), and Abberior STAR Red (goat-anti-Rabbit). Nuclear DNA was stained with DAPI (4’,6-diamidino-2-phenylindole) (1 mg/ml) (Sigma-Aldrich).
Alexa 680
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Alexa 680 is a fluorescent dye that can be used in various biomedical and life science applications. It has an excitation maximum of 679 nm and an emission maximum of 702 nm, making it suitable for detection in the near-infrared region of the spectrum.
Automatically generated - may contain errors
Lab products found in correlation
Irdye 800, by Rockland Immunochemicals (8 mentions)
Odyssey infrared imaging system, by LI COR (7 mentions)
Lsm 700, by Zeiss (4 mentions)
Odyssey infrared scanner, by LI COR (3 mentions)
Alexa fluor 647, by Thermo Fisher Scientific (2 mentions)
Nitrocellulose membrane, by Merck Group (2 mentions)
Alexa 800, by Thermo Fisher Scientific (2 mentions)
Xcell 2 blot module, by Thermo Fisher Scientific (2 mentions)
Texas red, by Thermo Fisher Scientific (2 mentions)
C57bl 6j, by Jackson ImmunoResearch (2 mentions)
Ab6556, by Abcam (2 mentions)
Hiv 1 env peptides, by Biomatik (2 mentions)
Poly ic lc, by Thermo Fisher Scientific (2 mentions)
Tissue tek oct, by Avantor (1 mentions)
Anti mhcii, by Thermo Fisher Scientific (1 mentions)
Anti inos, by Thermo Fisher Scientific (1 mentions)
Anti cd86, by Thermo Fisher Scientific (1 mentions)
Ivis spectrum imager, by PerkinElmer (1 mentions)
Nupage tris acetate gel, by Thermo Fisher Scientific (1 mentions)
Anti pgk1 antibody, by Thermo Fisher Scientific (1 mentions)
31 protocols using alexa 680
1
Comprehensive Immunolabeling Protocol for RNA Splicing
2
Immunophenotyping of Lymph Node Cells
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One set of lymph nodes (both sacral and axillary) from both groups were snap-frozen in Tissue-Tek OCT (VWR, Radnor, PA) and sectioned at the Winship Cancer Institute’s Pathology Core. Frozen sections of excised sacral and axillary nodes were blocked in 10% BSA in PBS and incubated with primary antibody overnight, and then secondary antibody for 2 h. Primary antibodies were anti-CD86 (Cat: MA1-10299), anti-iNOS (Cat: PA3-030A), anti-MHCII (Cat: MA5-16913) [all 3 from Thermo Fisher Scientific] and anti-IL12A (Acris Antibodies, San Diego, CA; Cat: AM32704AF-N). These sections were detected using secondary antibodies conjugated with Alexa Fluor 647 or Alexa 680 (Thermo Fisher Scientific) and imaged by confocal microscopy using a Zeiss LSM 700.
3
Subcutaneous Hydrogel-based Protein Delivery
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First, 7-week-old male C57BL/6 albino mice received a single subcutaneous injection of fluorescently labeled BSA or rhGALNS. The proteins were labeled with ALEXA 680 (ThermoFisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. Precursor solutions were prepared to reach a final concentration of 20% w/v four-arm PEG-Ac or eight-arm PEG-Ac + PEG-diSH, loaded with BSA or rhGALNS. Hydrogel mixtures (200 µL) were subcutaneously injected into the left flank of each mouse. The mice were anesthetized with isoflurane, and fluorescent images were acquired on an IVIS Spectrum imager (Perkin Elmer, Hongkong, China) every 24 h for up to 24 days.
All IVIS images were acquired under the same field of view using autoexposure. The data were analyzed using Living Image software (version 4.5). Fluorescent regions were quantified and expressed as Radiant Efficiency (p/s/sr)/(µW/cm2). Analyses were conducted with a Holm–Šídák’s multiple comparison test. p < 0.05 was considered statistically significant.
All IVIS images were acquired under the same field of view using autoexposure. The data were analyzed using Living Image software (version 4.5). Fluorescent regions were quantified and expressed as Radiant Efficiency (p/s/sr)/(µW/cm2). Analyses were conducted with a Holm–Šídák’s multiple comparison test. p < 0.05 was considered statistically significant.
4
SDS-PAGE and Immunoblotting Optimization
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Standard 10% to 14% SDS-PAGE gels were used for separation of most proteins except for HTT, which was better analyzed by NuPAGE Tris-Acetate gels from Invitrogen specially formulated for detection of proteins with large molecular weight. The boiled samples were separated on SDS-PAGE and transferred to nitrocellulose membranes from Millipore. After blocking with 5% nonfat milk in Tris-buffered saline with 0.1% Tween-20 for 1 hour, membranes were incubated with primary antibodies. Secondary antibodies conjugated with Alexa-800 or Alexa-680 (Invitrogen) or HRP (KPL) were used and the signals were detected by the Odyssey Infrared Imaging System and quantified by Odyssey Application Software 3.0 or by densitometry of the digital images using ImageJ software (NIH).
5
Western Blot Analysis of PKG1 Isoforms
6
Western Blot Analysis of Recombinant Proteins
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Proteins were extracted using the trichloroacetic acid method (51 (link)). Proteins were then separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (52 (link)) and transferred to nitrocellulose membranes using wet blotting (XCell II Blot Module, Invitrogen). Membranes were incubated overnight with primary anti-sfGFP antibodies (Abcam, ab6556) or anti-Pgk1 antibodies as loading control (ThermoFisher Scientific, Monoclonal antibody 22C5D8). Primary antibodies were detected using secondary antibodies labelled with Alexa 680 (Invitrogen, Germany) or IRDye800 (Rockland Immunochemicals Inc., USA). Detection was performed using an Odyssey Infrared Imaging Systems (Li-Cor Biosciences).
7
SDS-PAGE and Western Blot Analysis
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Standard 8% SDS-PAGE gels were used for separation of Yl-eGFP-3xHA protein. The boiled samples were separated on SDS-PAGE and transferred to nitrocellulose membranes from Millipore. After blocking with 5% nonfat milk in Tris-buffered saline with 0.1% Tween-20 for 1 hour, membranes were incubated with primary antibodies. Secondary antibodies conjugated with Alexa-800 or Alexa-680 (Invitrogen) were used and the signals were detected by the Odyssey Infrared Imaging System and quantified by Odyssey Application Software 3.0 or by densitometry of the digital images using ImageJ software (NIH).
8
In Vivo Structural Imaging of Mouse Vasculature
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For imaging of anesthetized animal, the mice were anesthetized using isoflurane (1–1.5 % in oxygen, maintaining a breathing frequency at 2 Hz) and placed on a heat blanket to maintain body temperature at 37.5 °C. Eye ointment was applied and the animal was place on a 3D motorized stage for navigation under the microscope. For structural imaging, vasculature of wild-type mice (N=3, 10–12 weeks, male, C57BL/6J, The Jackson Laboratories) was labeled through retro-orbital injection with fluorescein, Alexa 680, and Texas Red (25mg dextran conjugate dissolved in 200 μl sterile saline, 10kDa molecular weight, Invitrogen).
For awake imaging, the animal was fixed on a custom-made stereotaxic plate by attaching its head-bar to the metal holders. The body of the animal was further secured in a tube of slippery inner walls to reduce motion.
For awake imaging, the animal was fixed on a custom-made stereotaxic plate by attaching its head-bar to the metal holders. The body of the animal was further secured in a tube of slippery inner walls to reduce motion.
9
Quantitative Immunoblotting of Tagged Proteins
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Proteins were extracted from mid log phase cell cultures grown in the respective medium using the trichloroacetic acid (TCA) method (Knop et al., 1999 (link)). Proteins were then separated by SDS-PAGE as described (Laemmli, 1970 (link)) and transferred to nitrocellulose membranes using a semi-wet blotter (XCell II Blot Module; Invitrogen). Membranes were incubated overnight with primary anti-GFP antibodies (Abcam) or anti-PGK1 antibodies (Molecular Probes). Secondary antibodies were labeled with Alexa680 (Invitrogen) or IRDye800 (Rockland Immunochemicals). Detection and quantification was performed with an Odyssey Infrared Imaging System (Li-Cor Biosciences).
10
Western Blot Analysis of Shigella Effectors
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Proteins were transferred onto Immobilon FL (Millipore) membrane using a semi-dry method. Primary antibodies used were as follows: anti-IpaA mouse monoclonal, gift from Kirsten Niebuhr (3 (link)); anti-IpaB mouse monoclonal, named H16, gift from Armelle Phalipon (49 (link)); anti-IpaC mouse monoclonal, mixture of J22 and K24, gift from Armelle Phalipon (50 (link)); anti-IpaD rabbit polyclonal, gift from Claude Parsot (51 (link)) or as described in Cheung et al. (14 (link)); anti-IpgD mouse monoclonal, gift from Kirsten Niebuhr (3 (link)); and anti-MxiC rabbit polyclonal, raised against a fragment of MxiC containing residues 74–355 and an N-terminal His tag (12 (link)). Near-infrared fluorescent secondary antibodies (rabbit IgG raised in goat and coupled to Alexa680, Invitrogen; mouse IgG raised in goat and coupled to DyLight800, Pierce) were visualized and quantified on a LI-COR Odyssey imaging system.
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