Mice were anesthetized using isoflurane and secured on a warming imaging plate in a supine position. The medial or soleus region of the tibial bone was surgically exposed removing soft tissue. The bone was carefully thinned to approximately 200 μm thickness using a microdrill. The leg was imbedded in agarose to stabilize the area and create an immersion well for the microscope objective. Images were collected on an Olympus FV-1000MPE upright laser scanning microscope with 25X 1.05NA water immersion objective using a Spectra-physics DeepSee-MaiTai Ti:sapphire pulsed laser for excitation, with emission filters for the detection of GFP and rhodamine (Tomato or DsRed fluorescent proteins) and qdot 705 (Invitrogen). Images were analyzed on Volocity 6.3 (Improvision). Analysis of cell co-localization is included in Supplemental Experimental Procedures .
Qdot 705
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom
The Qdot 705 is a fluorescent nanocrystal produced by Thermo Fisher Scientific. It is designed for use in various life science applications that require fluorescence detection and imaging.
Automatically generated - may contain errors
Lab products found in correlation
Volocity 6, by PerkinElmer (2 mentions)
Qdot525, by Thermo Fisher Scientific (1 mentions)
Fitc dextran solution, by Merck Group (1 mentions)
Evans blue, by Merck Group (1 mentions)
Fv1000 mpe upright laser scanning microscope, by Spectra-Physics (1 mentions)
Lsm7 mp system, by Zeiss (1 mentions)
Eclipse te2000 u, by Nikon (1 mentions)
6 protocols using qdot 705
1
In Vivo Intravital Bone Imaging
2
In Vivo Intravital Imaging of Bone
3
Visualizing Blood Vessels and CTCs
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In order to visualize the blood vessels, either 0.5% of Evans blue (Sigma-Aldrich) in PBS solution or 40-kDa of fluorescein isothiocyanate (FITC)–dextran solution (Sigma-Aldrich) was injected through the tail vein. In order to label specific cells in circulation, streptavidin conjugated quantum dots (Qdot525, Qdot705) were purchased from Invitrogen. These quantum dots were further conjugated with biotinylated antibodies. The CD41 antibody was obtained from Abcam for the purpose of labeling platelets. In order to label subpopulations of CTCs, CD24 and 133, antibodies were purchased from Miltenyi Biotech (Auburn, CA, USA).
4
Intravital Imaging of Bone Microstructure
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Mice were anesthetized using isoflurane and secured on a warming imaging plate in a supine position. The medial or soleus region of the tibia was surgically exposed removing soft tissue. The bone was carefully thinned using a microdrill. Exposed tibia was mounted and immobilized with a metal plate, with an imaging window. All images were collected on an Olympus FV-1000MPE upright laser scanning microscope with a 25X 1.05 NA water immersion objective using a Spectra-physics DeepSee-MaiTai Ti:sapphire pulsed laser for excitation, with emission filters for the detection of second harmonic (compact bone), FITC (GFP), rhodamine (Tomato or DsRed fluorescent proteins) and qdot 705 (Invitrogen). To visualize mCherry expression, Ti-Sapphire laser excitation was tuned to 740nm and emission collected by non-descanned PMT using a 595-645nm filterset.
5
Intravital Multiphoton Microscopy for In Vivo Imaging
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Intravital multi-photon microscopy studies were carried out using a Zeiss LSM7 MP system equipped with a tuneable titanium:sapphire solid-state two-photon excitation source (4W, Chameleon Ultra II, Coherent Laser Group) coupled to an Optical Parametric Oscillator (Chameleon Compact OPO; Coherent). Movies were acquired for 10 to 15 min with an X:Y pixel resolution of 512 × 512 in 2 μm Z increments producing up to 40 μm stacks. 3D tracking was performed using Volocity 6.1.1 (Perkin Elmer, Cambridge, UK). Values representing the mean velocity, displacement and meandering index were calculated for each object. Mice were anaesthetised IP using medetomidine (Domitor 0.5 mg/kg) and ketamine (50 mg/kg) and placed on a heated stage. Following removal of hair with a depilatory cream, dorsal skin was imaged. An intravenous injection of non-targeted quantum dots (Qdot705) (Life Technologies, UK) prior to imaging allowed visualisation of blood vessels.
6
Nasal Quantum Dot Deposition in Mice
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Six to eight week old female BALB/c mice (Harlan, Frederick, MD) were given a 46 nM quantum dot (20 nm Qdot 705, Life Technologies, Grand Island, NY) solution by either a 4-h nanoaerosol spray or a single 40 μL intranasal dose. Controls received 40 μL of PBS intranasally. Mice rested for 2 h before being administered a ketamine–xylazine cocktail. While under anesthesia, mice were euthanized and lungs were perfused in situ with 10 mL of PBS followed by 20 mL of 4 % depolymerized paraformaldehyde. Lungs were harvested and underwent cryosectioning (10 μm thick sections with Thermo Scientific HM550 cryostat, Waltham, MA) and imaged using a confocal microscope (Nikon Eclipse TE2000-U, Melville, NY).
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