Protein lysates were diluted using protein lysis puffer (2 µg/µl). After adding Tween 20 (0.05%, v/v) protein lysates were printed onto nitro-cellulose coated glass slides (Oncyte Nitrocellulose Film Slides, Grace Bio-Labs, Blend, OR, USA) using the Aushon 2470 solid-pin tool arrayer (Aushon Biosystems, Billerica, MA, USA). Antibody incubation and antibody-mediated signal amplification were performed as described [18] (link). Slides were scanned with the Odyssey NIR scanner (LI-COR Biosciences, Bad Homburg, Germany). Image analysis was carried out with GenePix-Pro 6.0 (Axon Instruments, Sunnyvale, USA). Data sets were analyzed using the RPPAnalyzer package [19] (link). Quantification results were normalized to the Fast Green FCF staining of total proteins as well as to the median antibody binding signal levels [20] (link).
Odyssey nir scanner
Manufactured by LI COR
Sourced in Germany
The Odyssey NIR scanner is a near-infrared (NIR) imaging system designed for quantitative Western blot and fluorescence analysis. It utilizes two-color detection to simultaneously capture signals from two different fluorescent dyes or labels. The Odyssey scanner provides high-sensitivity detection and accurate quantification of protein and nucleic acid samples.
Automatically generated - may contain errors
Lab products found in correlation
To pro 3, by Thermo Fisher Scientific (2 mentions)
Jpm oet, by MedChemExpress (2 mentions)
Gapdh, by Santa Cruz Biotechnology (2 mentions)
Ventana benchmark xt, by Roche (2 mentions)
Protein assay, by Bio-Rad (2 mentions)
Oncyte nitrocellulose film slides, by Grace Bio-Labs (1 mentions)
Genepix pro 6, by Molecular Devices (1 mentions)
Iblot system, by Thermo Fisher Scientific (1 mentions)
Nupage novex bis tris gel, by Thermo Fisher Scientific (1 mentions)
Hrp linked secondary antibody, by Agilent Technologies (1 mentions)
Odyssey fc system, by LI COR (1 mentions)
Ab52903, by Abcam (1 mentions)
Ab6046, by Abcam (1 mentions)
Nupage novex 4 12 bis tris gel, by Thermo Fisher Scientific (1 mentions)
Odyssey blocking buffer, by LI COR (1 mentions)
11 protocols using odyssey nir scanner
1
Quantitative Protein Profiling by RPPA
2
Protein Extraction and Quantification
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For total protein extracts, 8 M urea lysis buffer was used (8 M urea, 0.5% [v:v] Triton X-100 [Sigma-Aldrich, X100], DTT [100 mM], Complete® protease inhibitor [PI; Roche, 11873580001]), long with phosphatase inhibitor cocktail (PIC) 2 and 3 (Sigma, P5726 and P0044). Protein concentration was determined by Bio-Rad protein assay (Bio-Rad 500–0006). Proteins (50 µg) were separated using NuPAGE® Novex® Bis-Tris Gels (Invitrogen, NP0322, NP0321, WG1403, WG1402A), transferred to nitrocellulose membranes using Invitrogen's iBlot system. Bound antibodies were imaged using appropriate near-infrared (NIR) dye conjugates (Li-Cor) and an Odyssey NIR scanner (Li-Cor Biosciences) or using HRP-linked secondary antibodies (Dako) and SuperSignal West Femto substrate (Pierce, 34096) with a Li-Cor Odyssey Fc System. Image studio 3.1 was used for quantification. For statistical assessment, mean intensity values were used, while bar graphs of quantifications show fold changes.
3
Evaluating cRGD-ZW800-1 Binding Capacity in U-87 MG Cells
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To evaluate the binding capacity of cRGD-ZW800-1, U-87 MG cells were plated in a 96-well plate at a density of ∼40,000 cells per well. At 90-100% confluence, cells were washed and incubated with various concentrations of cRGD-ZW800-1 at 4°C or 37°C for 1 h (binding assay). cRGD-ZW800-1 was added to the U-87 MG cells in various concentrations: 0, 125, 250, 500, and 1000 nM.
For in vitro competition, U-87 MG (αvβ3 and αvβ5 positive, αvβ6 negative) and HT-29 (αvβ3 negative, αvβ5 and αvβ6 positive) cells were plated in a 96-well plate at a density of ∼40,000 and ∼60,000 cells per well, respectively. Subsequently, 500 nM of cRGD-ZW800-1 was simultaneously added to the cells and incubated at 37°C for 2 h. At the same time, half of the cells were also incubated with 500 nM unlabeled (“cold”) cRGD.
Both the cells in the binding and the competition experiment were then washed twice and imaged with the Odyssey NIR scanner (LI-COR Biosciences, Lincoln, Nebraska: focus offset 3 mm; 800-nm channel). Next, cells were permeabilized with a 40/60 mixture of acetone and methanol followed by a washing step and a 5 min incubation with ToPro3 (1/2000, Invitrogen), a far red fluorescent dye (642/661nm). The wells were then washed and again imaged with the Odyssey scanner (focus offset 3 mm; 700-nm channel) to quantify the number of cells in each well. The experiments were performed in triplicate.
For in vitro competition, U-87 MG (αvβ3 and αvβ5 positive, αvβ6 negative) and HT-29 (αvβ3 negative, αvβ5 and αvβ6 positive) cells were plated in a 96-well plate at a density of ∼40,000 and ∼60,000 cells per well, respectively. Subsequently, 500 nM of cRGD-ZW800-1 was simultaneously added to the cells and incubated at 37°C for 2 h. At the same time, half of the cells were also incubated with 500 nM unlabeled (“cold”) cRGD.
Both the cells in the binding and the competition experiment were then washed twice and imaged with the Odyssey NIR scanner (LI-COR Biosciences, Lincoln, Nebraska: focus offset 3 mm; 800-nm channel). Next, cells were permeabilized with a 40/60 mixture of acetone and methanol followed by a washing step and a 5 min incubation with ToPro3 (1/2000, Invitrogen), a far red fluorescent dye (642/661nm). The wells were then washed and again imaged with the Odyssey scanner (focus offset 3 mm; 700-nm channel) to quantify the number of cells in each well. The experiments were performed in triplicate.
4
Quantifying Binding Capacity of Tracer Conjugates
5
Activin A-Induced SMAD3 Phosphorylation
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HEK293 autophagy reporter cells were exposed to 50 μg/mL recombinant activin A for 5 min at the end of 2 h incubation in normal growth medium containing 0.1% FBS. Cell were lysed by cell scraping on ice in a buffer containing 8 M urea, 0.5% (v/v) Triton X‐100, 100 mM DTT, 1xComplete® protease inhibitor, and 8% phosphatase inhibitor cocktail I and III (Sigma). Protein concentration was determined by BioRad protein assay (BioRad). Equal amounts of proteins were separated using NuPAGE® Novex® 4‐12% Bis‐Tris Gels (Invitrogen) and dry blotted on nitrocellulose membranes. The membrane was blocked, and antibodies were diluted in a 1:1 mixture of Odyssey blocking buffer (Li‐Cor) and TBST (20 mM Tris, pH 7.6, 137 mM NaCl with 0.1 % Tween 20). Bound antibodies were imaged by near infrared fluorescence using appropriate fluorescent dye labelled secondary antibodies and an Odyssey NIR scanner (Li‐Cor Biosciences). Images were processed using the Li‐Cor Odyssey software image studio 2.0. The antibodies used for immunostaining were pSMAD3 (Ser423/Ser425) (Abcam, ab52903), diluted 1:1000, and beta‐tubulin (Abcam, ab6046), diluted 1:5000.
6
Evaluating VGT-309 Binding Specificity
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To evaluate the binding specificity of VGT-309 to individual cysteine cathepsins as well as the activity dependence of the probe, we conducted fluorescent SDS-PAGE analysis of probe-labeled species using a protocol previously described [25 (link)]. Briefly, we incubated OE19 cells with 1 μM VGT-309 for 2 hours after pretreatment of cells for 30 minutes with 100 μM JPM-OEt (MedChemExpress, Monmouth, NJ), a pan-cathepsin inhibitor. We lysed the cells and loaded lysates and a fluorescent molecular weight maker on an SDS-PAGE gel and ran the gel for 15 minutes at 80 V and subsequently at 130 V until the dye front had run off the gel. Gels were imaged on the Odyssey NIR scanner (LI-COR Biosciences, Lincoln, NE) and individual cathepsin bands were assigned by molecular weight. To control for equal protein loading, gels protein bands were transferred to a PVDF membrane and probed for GAPDH (Santa Cruz Biotechnology) as a loading control.
7
Tumor Tissue Analysis via Fluorescence Imaging
8
EGFR and Ki-67 Expression in Tumor Tissues
9
Multimodal Tumor Imaging and Pathology
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Formalin-fixed paraffin-embedded tumor tissues were sectioned at 5 µm thickness and fluorescence imaging was performed using the Odyssey NIR scanner (Li-COR Biosciences). In addition to being scanned using the Odyssey NIR scanner, all histologic sections were stained with standard hematoxylin–eosin immunohistochemical staining (H&E) so that representative tissue sections from each specimen could be assessed by a neuropathologist for tumor status—the current gold standard for tumor determination. To confirm the presence of EGFR, additional sections were stained with immunohistochemistry (IHC) for anti-human EGFR (predilute, rabbit, clone 5B7, 790-4347, Ventana, Tucson, AZ). Automated immunohistochemical staining was performed with Ventana Benchmark XT (Ventana Medical Systems, Inc., Tucson, AZ, USA).
10
Evaluating Cysteine Cathepsin Binding Specificity
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