The polyplexes and amine-reactive Alexa Fluor 750 dye were dissolved in 0.1 M sodium bicarbonate buffer (0.5 mg/mL) and DMSO (10 mg/mL), respectively. Then, the polyplex solution was mixed with dye solution at a volume ratio of 1:10. The reaction was conducted for 1 h at room temperature under magnetic stirring. The unconjugated dye was eliminated from the solution by ultrafiltration in an Amicon cell (regenerated cellulose membrane, MWCO = 5 kDa). Then, the polyplex solution was washed several times with PBS (pH 7.4) until no fluorescence absorption of Alexa Fluor 750 was detectable in the filtrate. Before imaging study, the mice were anesthetized by intraperitoneal injection of 150 mL 4% chloralic hydras. Optical imaging was performed using an in vivo fluorescence imaging system (Carestream, USA) with emission at 720 nm and excitation at 790 nm after the mice were injected with the polyplex solutions. The fluorescence images were captured at different time points. The fluorescence intensity was quantified using Carestream MI software. Ex vivo imaging study of major organs and tumors were performed as well under the same image acquisition conditions.
Mi software
Manufactured by Carestream
Sourced in United States
Carestream MI software is a comprehensive imaging and informatics platform designed for medical and dental laboratories. It provides advanced tools for managing, storing, and analyzing digital images and patient data. The software's core function is to facilitate efficient image acquisition, archiving, and retrieval, enabling healthcare professionals to streamline their workflow and enhance patient care.
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Lab products found in correlation
Dnmt1, by Abcam (2 mentions)
β actin cat a 5316, by Merck Group (2 mentions)
β actin, by Carestream (2 mentions)
Gradient acrylamide gel, by Bio-Rad (2 mentions)
Anti mouse igg, by Thermo Fisher Scientific (2 mentions)
Superblock tbs blocking buffer, by Thermo Fisher Scientific (2 mentions)
Iblot, by Thermo Fisher Scientific (2 mentions)
Hrp conjugated anti rabbit igg, by Thermo Fisher Scientific (2 mentions)
Nupage 4 12 bis tris gel, by Thermo Fisher Scientific (2 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (2 mentions)
Pierce bca protein assay, by Thermo Fisher Scientific (2 mentions)
In vivo fx pro, by Bruker (1 mentions)
Osteosense 680ex, by PerkinElmer (1 mentions)
Small animal in vivo fluorescence imaging system, by Carestream (1 mentions)
In vivo imaging system fx pro, by Kodak (1 mentions)
Fx pro, by Carestream (1 mentions)
Sc 6236, by Santa Cruz Biotechnology (1 mentions)
Sc 481, by Santa Cruz Biotechnology (1 mentions)
Anti rabbit or mouse horseradish peroxidase conjugated secondary antibodies, by Santa Cruz Biotechnology (1 mentions)
Gapdh sc 25778, by Santa Cruz Biotechnology (1 mentions)
13 protocols using mi software
1
Fluorescent Polyplex Characterization
2
Protein-Protein Interaction Screening
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For western assays, cells were lysed in modified kinase lysis buffer (KLB) (Ditlev et al., 2012 (link)) as previously described, with 0.1% SDS added to aid in solubilizing large OptoEphB2 or optoEphB2-KD clusters. SH2 domain rosette screening was performed as described previously (Machida et al., 2007 (link)). Briefly, lysates were diluted with 2× spotting solution (100 mM Tris–HCl, pH 6.8, 30% glycerol, 2% SDS) to approximately 4 μg/μl and spotted in duplicate in a rosette pattern on nitrocellulose membranes. Membranes were blocked with 5% milk in TBST [25 mM Tris– HCl, pH 8.0, 150 mM NaCl, and 0.05% (v/v) Tween-20] and incubated with 200 nM GST-SH2 domains labeled with GSH-HRP for 2 h in a 96-well chamber plate. Each well was washed with TBST and chemiluminescent detection and quantification was performed using Carestream Image station system and Carestream MI software.
3
Establishing and Monitoring Ovarian Cancer Xenografts
4
In Vivo Bone Growth Imaging
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In vivo bone growth were imaged and analyzed with two commercial fluorescent in vivo bisphosphonate imaging probe OsteoSense® 680 Ex (OS680) and OstenSense® 800 (OS800) (PerkinElmer, MA, USA) with two distinct wavelengths modified from suggested protocol and previous study [23 (link)]. Briefly, anesthetized mice were injected retro-orbitally with dissolved fluorescent probes (10 nmol/kg) 48 h before imaging. Hair was removed from the hind legs. Florescent signals and X-ray images were captured by combination of multispectral fluorescence and digital radiograph function of in vivo Multispectral FX PRO system. After anesthesia, signals of injected Os680 were detected in vivo with excitation wavelength at 650 nm and emission at 700 nm at week 2, while Os800 was injected and detected (excitation 760 nm/emission 830 nm) at week 3. At week 4, both florescent signal of Os650 and Os800 in both sides of femur were measured ex vivo after femurs were harvested. Florescent and radiographic images obtained were overlayed and analyzed with Carestream MI Software. ROIs of same area were selected from operated and contralateral femurs versus surrounding skin (as background signal). Os signal intensity of fractured right femurs was normalized by intensity of contralateral side.
5
In vivo Fluorescence Imaging of Cy7.5-Loaded Micelles
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Cy7.5 instead of Cy3 was loaded into PEG‐PEI micelle for fluorescence imaging in the same way as that for preparation of Cy3‐loaded micelles. Before imaging study, the rats of different groups were anesthetized by intraperitoneal injection of pentobarbital sodium (50 mg kg−1). Optical imaging was performed using a small animal in vivo fluorescence imaging system (Carestream, USA) with emission at 720 nm and excitation at 790 nm after the mice were injected with the polyplex solutions. The fluorescence images were captured at different time points. The fluorescence intensity was quantified using Carestream MI software. Ex vivo imaging study of carotid was performed as well under the same image.
6
Visualizing Visual System Degeneration
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To detect the connection and degeneration of the visual system from the eye to the LGN and SC, dyes were injected as previously described. 20, 35 In brief, 2 ll of freshly prepared 3% rhodamine-b-isothiocyanate (RITC) was injected intravitreally with a 28-gauge needle at the pars plana of each right eye. The brains were removed 3 days after injection. The differences in LGN and SC staining were macroscopically observed in the low-CSFp, high-IOP, and anesthesia control groups by using the Kodak In-Vivo Imaging System FX Pro (n ¼ 5 per group). Fluorescence intensity in the left LGN and SC of low-CSFp and high-IOP brains were compared to those in the anesthesia control group. The relative average value of RITC fluorescence brightness in the LGN and SC was analyzed with the Carestream MI software.
7
In vivo NIR Imaging of Arthritis
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At 2 weeks after secondary immunization, radiographic imaging (X-ray) was performed to assess the joints, thus determining no bone destruction and arthrostenosis in CIA models. Firstly, mice with two symptomatic paws were selected for NIR fluorescence imaging to demonstrate the availability of the imaging modes by iLPs. Mice were intravenously injected with LPs or iLPs, respectively. The equivalent ICG dose was kept at 0.5 mg/kg. The NIR images of mice were obtained at predetermined time points (15 min, 1 h, 3 h, 6 h, 12 h, 24 h) using the ex/in vivo imaging system (Carestream FX Pro; Carestream Health Inc., Toronto, CA, USA) with a 704 nm excitation wavelength and 745 nm filter. Then, mice with one symptomatic paw were selected to demonstrate the accuracy of the imaging modes by iLPs. Then, mice were intravenously injected with iLPs, LPs, or free ICG, respectively. The NIR images of mice were also obtained at predetermined time points (15 min, 2 h) using the ex/in vivo imaging system. At 24 h after injection, the mice were sacrificed and main organs including heart, liver, spleen, lung, and kidney were harvested. Then, main organs were photographed to analyze the biodistribution of iLPs or LPs using the ex/in vivo imaging system. All semi-quantitative data was obtained by Carestream MI software (Carestream Health Inc.).
8
Cardamonin Modulates Cellular Proteins in HCT116 Cells
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HCT116 cells (2×106 cells/well) were cultured overnight in a 6-well plate and treated with cardamonin (0, 20, 40 and 80 µM) for 72 h. Cell lysates were prepared by the addition of 100 µl lysis buffer for 15 min. Protein content was determined using the BCA method. A total of 50 µg protein per lane was separated by 8–10% SDS-PAGE and electrotransferred onto polyvinylidene difluoride membranes. The membranes were blocked with TBST containing 5% non-fat dried milk at 37°C for 1 h and incubated with specific primary antibodies targeted at B-cell lymphoma-associated X (Bax, sc-6236, 1:1,000), Myc proto-oncogene protein (c-MYC, sc-789, 1:1,000), octamer-binding transcription factor 4 (Oct4, sc-9081, 1:1,000), and cyclin E (sc-481, 1:1,000) (all from Santa Cruz Biotechnology, Inc.), testes-specific protease 50 (TSP50, ab181993, 1:1,000; Abcam), NF-κB (sc-101749, 1:1,000) and GAPDH (sc-25778, 1:1,000) (both from Santa Cruz Biotechnology, Inc.) overnight at 4°C. Following three washes with TBST, membranes were incubated with anti-rabbit or mouse horseradish peroxidase-conjugated secondary antibodies (sc-2004 or sc-2005, 1:5,000; Santa Cruz Biotechnology, Inc.) for 1 h at room temperature. Signals were detected by enhanced chemiluminescence (ECL Plus detection system) and band density was analyzed using Carestream MI software (Carestream Health, Inc., Rochester, NY, USA).
9
Optimized Immunoblotting for Protein Quantification
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Immunoblotting was performed on total homogenates as described previously (Matosin et al., 2013) , with minor modifications optimised for the measurement of each protein of interest. Each lane was loaded with 5 μg (human) or 10 μg (animal) of total protein. These protein concentrations were determined to be in the linear range of detection (data not shown). Relative protein densities were determined by immunoblot analyses using polyclonal antibodies as previously reported: mGluR5
[ABCAM ab27190; human 1:250, rat 1:500 (Matosin et al., 2013) ], Norbin [ABCAM ab130507;
human and rat 1:500], and Tamalin [ABCAM ab30576; human 1:100, rat 1:1000 (Tai et al., 2010) ].
mGluR5 monomer was detected at 135 kDa whilst the mGluR5 dimer was detected at 270 kDa.
Individual bands were totalled before normalisation to respective ß-actin and pooled samples to gain a measure of total mGluR5. Samples were visualised using an enhanced chemiluminescent detection kit (Bio-Rad). Band density was detected by the Gel Doc 2200 Pro (Carestream Molecular Imaging, USA) and quantified with Carestream MI software (v 5.0.4.44, Carestream Molecular Imaging). All bands were within the limits of saturation. Protein measures were subsequently normalised to their respective ß-actin density. Experiments and quantification were performed blind to diagnosis (human)
or treatment group (rat).
[ABCAM ab27190; human 1:250, rat 1:500 (Matosin et al., 2013) ], Norbin [ABCAM ab130507;
human and rat 1:500], and Tamalin [ABCAM ab30576; human 1:100, rat 1:1000 (Tai et al., 2010) ].
mGluR5 monomer was detected at 135 kDa whilst the mGluR5 dimer was detected at 270 kDa.
Individual bands were totalled before normalisation to respective ß-actin and pooled samples to gain a measure of total mGluR5. Samples were visualised using an enhanced chemiluminescent detection kit (Bio-Rad). Band density was detected by the Gel Doc 2200 Pro (Carestream Molecular Imaging, USA) and quantified with Carestream MI software (v 5.0.4.44, Carestream Molecular Imaging). All bands were within the limits of saturation. Protein measures were subsequently normalised to their respective ß-actin density. Experiments and quantification were performed blind to diagnosis (human)
or treatment group (rat).
10
Western Blot Quantification Protocol
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