smFISH probes (32 probes per gene) were designed using the Stellaris Probe Designer (Biosearch Technologies) and ordered as unlabelled DNA oligos. Labelling was done according to Gaspar et al, 2017 (link) (Gaspar et al, 2017 (link)). Briefly, unlabelled ddUTP was conjugated to an ATTO dye NHS ester (ATTO565 or ATTO633, Atto-tec), then the labelled ddUTP was added to the 3’-end of each probe with terminal deoxynucleotidyl transferase. Probes were purified by ethanol precipitation.
Atto633
Manufactured by ATTO-TEC
Sourced in Germany
ATTO633 is a fluorescent dye developed by ATTO-TEC for use in various analytical and imaging applications. It has an absorption maximum at 633 nm and an emission maximum at 657 nm, making it suitable for detection and labeling in the red spectral region.
Automatically generated - may contain errors
Lab products found in correlation
Stellaris probe designer, by Biosearch Technologies (1 mentions)
Atto 565, by ATTO-TEC (1 mentions)
6 hydroxy 2 5 7 8 tetramethylchroman 2 carboxylic acid trolox, by Merck Group (1 mentions)
Benzonase nuclease, by Merck Group (1 mentions)
Sodium hydroxide (naoh), by Merck Group (1 mentions)
Atto488, by ATTO-TEC (1 mentions)
Teneo, by Thermo Fisher Scientific (1 mentions)
Tecnai, by Thermo Fisher Scientific (1 mentions)
Zetasizer nano zsp, by Malvern Panalytical (1 mentions)
Eclipse ti, by Nikon (1 mentions)
Peg biotin, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Pd 10 column, by GE Healthcare (1 mentions)
4 protocols using atto633
1
Single-Molecule FISH Probe Labeling
2
Single-Molecule Dye Labeling Protocol
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Boric acid and DMSO (both pro analysis) were obtained from Fluka (Steinheim, Germany) and sodium hydroxide (≥99 %) from Merck (Darmstadt, Germany). Atto 488, Atto 495 and Atto 633 were from Atto Tec (Siegen, Germany). The three individual fluorophore molecules were dissolved in DMSO (at 2.5, 3.3 and 2.5 mM, respectively) and further diluted as described below. Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) and Benzonase nuclease (ultrapure grade) were purchased from Sigma Aldrich (Steinheim, Germany). Water (18.2 MΩ cm resistivity at 25 °C) was from a Millipore (Billerica, MA, USA) apparatus.
3
Multimodal Characterization of Mesoporous Silica Nanoparticles
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Morphological characterization of the synthesized MSNs was performed by transmission electron microscopy (TEM) using an FEI Tecnai and scanning electron microscopy (SEM) using an FEI Teneo in the secondary electron mode. Size and electrokinetic potential (ζ-potential) were measured by dynamic light scattering at 25°C at an angle of 90° using the Zetasizer Nano ZSP (Malvern Instruments, Malvern, UK). Fluorescence microscopy (Nikon Eclipse Ti) was used to evaluate dual labeling by two probes: Atto 633–maleimide (Atto-Tec, Siegen, Germany) and FITC-NHS ester for MSNs and Atto 633–maleimide and DOPC:PC TopFluor 488 (99.96:0.04) for MSN-Lip.
4
Protein Cross-Linking and Purification
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Protein pellets were dissolved in 8 M guanidinium HCl (Thermo Scientific). For cross-linking reactions, a tenfold molar excess of maleimide-modified label (Alexa 488 Fluor (Thermo Scientific, Cat# A10254), Alexa 594 Fluor (Thermo Scientific, Cat# A10256), ATTO633 (ATTO-TEC GmbH, Cat# AD 633-41), or PEG-biotin (Thermo Scientific, Cat# 21911)) was added. The samples were incubated for 2 to 24 h at 22 °C. To remove excess label and/or denaturant, the samples were passed over PD-10 columns (GE Healthcare) equilibrated with assembly buffer (10 mM HEPES pH 7.4, 100 mM NaCl, 0.1 mM NaN3). Protein concentrations were determined using the bicinchoninic acid assay (BCA, Pierce).
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