HEp-2 cells expressing GalT-GFP-SNAP or ER-GFP-SNAP were treated with FDG for 4 h prior to incubation with Alexa555 labeled Stx1-mut-BG for 1 h (toxin labeling was performed using Alexa Fluor® 555 Microscale Protein Labeling Kit, Molecular Probes®). To allow Stx transport to the ER, HEp-2-ER-GFP-SNAP cells were washed and incubated with fresh complete medium for additional 4 h. The cells were fixed with 10% (w/v) formalin-solution for 15 minutes, followed by permeabilization in 0.1% (w/v) Triton X-100 for 5 minutes at room temperature. The cells were incubated in blocking solution (5% (v/v) FBS in PBS) for 30 minutes, before incubation with the appropriate primary and secondary antibodies. The cells were mounted in Prolong®Gold containing DAPI (Molecular Probes®), and imaged on a laser scanning confocal microscope LSM 780 (Carl Zeiss), equipped with a 63x objective, NA 1.4. Images were prepared using ImageJ software [55 (link)].
Alexa fluor 555 microscale protein labeling kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Alexa Fluor® 555 Microscale Protein Labeling Kit is a laboratory tool designed for covalent labeling of proteins with the Alexa Fluor® 555 dye. The kit includes the necessary components to facilitate the efficient and site-specific conjugation of the fluorescent dye to proteins in a microscale format.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 780, by Zeiss (2 mentions)
Su5000, by Leica (2 mentions)
Alexa fluor 488 microscale protein labeling kit, by Thermo Fisher Scientific (2 mentions)
Prolong gold containing dapi, by Thermo Fisher Scientific (1 mentions)
Provis bx51 microscope, by Hamamatsu Photonics (1 mentions)
Axio observer widefield microscope, by Zeiss (1 mentions)
Alexa fluor 594 goat anti rabbit antibody, by Thermo Fisher Scientific (1 mentions)
Tamoxifen tam, by Merck Group (1 mentions)
Stx2 holotoxin, by BEI Resources (1 mentions)
Stx2b, by BEI Resources (1 mentions)
Anti gm130, by BD (1 mentions)
Alexa fluor 488 goat anti mouse, by Thermo Fisher Scientific (1 mentions)
Axiovert, by Zeiss (1 mentions)
Mouse anti α actinin antibody, by Merck Group (1 mentions)
8 protocols using alexa fluor 555 microscale protein labeling kit
1
Visualizing Shiga Toxin Transport in Cells
2
Fluorescent Labeling of rTvα-actinin 2
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To estimate the binding of rTvα-actinin 2 to the host cells, Tvαactinin 2 proteins were labeled with a fluorescent probe using an Alexa-Fluor 555 Microscale Protein Labeling Kit (A30007; Molecular Probes, USA). One hundred microgram of recombinant polypeptides was reacted with Alexa-Fluor 555 succinimidyl ester, and then passed through the spin-filters supplied with the kit to remove unincorporated probe. Fluorescence of the proteins was measured at 280 and 555 nm in order to check labeling of the protein as directed by the manufacturer. Alexa-Fluor 555-labeled α-actinin 2 (100 μg/ml) was stored at -20ºC before being used for the binding assays.
3
Imaging Keratinocyte Morphology and Internalization
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Keratinocytes were fixed with 3% paraformaldehyde for 10 minutes. When nuclei staining was required, cells were incubated with DAPI (4′,6-diamidino-2-phenylindole) for 15 min and cells were mounted in glass slides using Mowiol. Images from necrosis and autophagy assays were acquired in Olympus Provis BX51 microscope coupled to monochromatic camera SPOT RT using SimplePCI 6 software (Hamamatsu, Japan). For cell morphology, phase contrast images were acquired with 10X objective and for video time-lapse acquisition, real time phase contrast images were acquired every 5 min, during 6 hours with 40X objective in Widefield Zeiss Axio Observer microscope, using Zen acquisition software (Carl Zeiss AG, Oberkochen, Germany). For LAAO internalization analysis, LAAO and Bovine Serum Albumin (BSA) labeling was performed using Alexa Fluor® 555 Microscale Protein Labeling Kit (ThermoFisher Scientific, Leicestershire, United Kingdom) as per manufacturer’s instructions. Images were acquired using 60X objective in Widefield Zeiss Axio Observer microscope, using Zen acquisition software (Carl Zeiss AG, Oberkochen, Germany).
4
Labeling TRPV1 Antibodies for OSC Imaging
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Rabbit anti TRPV1 antibody (25 μg; Synaptic Systems, no. 444 003) were labeled with Alexa Fluor 555 Microscale Protein Labeling Kit (Thermo Fisher Scientific, no. A30007) according to the manufacturer’s description but using the recommended 10× dye/protein ratio to ensure labeling despite the presence of BSA. OSCs were incubated with labeled antibody (25 μg/ml) for 2 hours or, alternatively, unlabeled antibody (10 μg/ml) for 90 min followed by washing three times for 15 min (recording solution) and 90-min incubation with a 1:200 dilution of Alexa Fluor 594 goat-anti-rabbit antibody (Invitrogen, no. A-11012). Antibodies were all incubated in medium at 37°C under rotation, and nPDs were added 1 hour before the end of the incubation. Once nPDs had been added, the slices were kept in the incubator without agitation to favor nPD penetration in the tissue.
5
Fluorescent Labeling of Shiga Toxin 2B
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We have previously described the GFP-SNX1 and GFP-Rab5 plasmids used in this study [16 (link)]. Monoclonal anti-GM130 (#610822) was from BD Biosciences (San Jose, CA, USA). Tamoxifen (TAM) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Derivative compounds were synthesized at the Center for Innovative Drug Discovery (University of Texas at San Antonio, San Antonio, TX, USA; see Supplementary Materials ). All compounds were dissolved in DMSO and used at 10 µM final concentration unless specified otherwise.
STx2 holotoxin and STx2B were obtained from BEI Resources (Manassas, VA, USA). For transport assays, STx2B was labeled using Alexa Fluor™ 555 Microscale Protein Labeling Kit using manufacturer’s instructions (ThermoFischer, Waltham, MA, USA).
STx2 holotoxin and STx2B were obtained from BEI Resources (Manassas, VA, USA). For transport assays, STx2B was labeled using Alexa Fluor™ 555 Microscale Protein Labeling Kit using manufacturer’s instructions (ThermoFischer, Waltham, MA, USA).
6
Platinum Nanoparticles Electrodeposition and Protein Labeling on LIG Electrodes
7
Fluorescence Imaging of Neonatal Cardiomyocytes
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Neonatal cardiomyocytes were exposed to synthetic CPP-Ts or to the CPP-Ts sub peptide14–39 marked with Alexa Fluor 555 Microscale Protein Labeling Kit (555/565 nm; Invitrogen A30007, MA, USA). Confocal immunofluorescence was performed as previously described68 (link), using mouse anti-α-actinin antibody 1:150 (Sigma-Aldrich A7811, MO, USA), goat anti-mouse Alexa Fluor 488 1:500 (Invitrogen R37120, MA, USA), and TO-PRO-3 probe 1:800 (Invitrogen T3605, MA, USA) for nuclear staining. For the sub peptide14–39 internalization assay in various cell lines, only TO-PRO-3 probe was used for nuclear staining.
Images were collected in a Zeiss Axiovert (Zeiss, CA, USA) confocal microscope. Three lasers were utilized, as follows: excitation at 488 nm and emission at 505–550 nm for Alexa 488; excitation at 568 nm and emission at 585–615 nm for Alexa 555; and excitation at 633 nm and emission at 650 nm for TO-PRO-3.
Images were collected in a Zeiss Axiovert (Zeiss, CA, USA) confocal microscope. Three lasers were utilized, as follows: excitation at 488 nm and emission at 505–550 nm for Alexa 488; excitation at 568 nm and emission at 585–615 nm for Alexa 555; and excitation at 633 nm and emission at 650 nm for TO-PRO-3.
8
Platinum Nanoparticles Electrodeposition and Protein Labeling on LIG Electrodes
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In order to demonstrate the platinum nanoparticles electrodeposition onto the LIG electrodes, a scanning electron microscopy analysis was performed using a scanning electron microscope SU5000 under the following conditions: 5 kV, 40 spot size, 50 Pa low vacuum, and magnification of 0.5, 2.5, 5, and 15 k.
To corroborate the presence and distribution of biotinylated hACE2 and recombinant S protein-RBD onto the LIG-nPt electrode surface, confocal microscopy images were taken on a Leica SPE confocal (Leica Microsystems, Wetzlar, Germany) at the Clemson Light Imaging Facility (Clemson Division of Research, Clemson University, Clemson SC, US). Biotinylated hACE2 was directly labeled with AlexaFluor™ 488 Microscale Protein Labeling Kit (Invitrogen™, green, λ 495/519 nm). Recombinant S protein-RBD was directly labeled with AlexaFluor™ 555 Microscale Protein Labeling Kit (Invitrogen™, orange/red, λ 555/565 nm).
To corroborate the presence and distribution of biotinylated hACE2 and recombinant S protein-RBD onto the LIG-nPt electrode surface, confocal microscopy images were taken on a Leica SPE confocal (Leica Microsystems, Wetzlar, Germany) at the Clemson Light Imaging Facility (Clemson Division of Research, Clemson University, Clemson SC, US). Biotinylated hACE2 was directly labeled with AlexaFluor™ 488 Microscale Protein Labeling Kit (Invitrogen™, green, λ 495/519 nm). Recombinant S protein-RBD was directly labeled with AlexaFluor™ 555 Microscale Protein Labeling Kit (Invitrogen™, orange/red, λ 555/565 nm).
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