On p18 and p26, all animals were anesthetized using intraperitoneal ketamine and dexmedetomidine (75/0.5 mg/kg). Tropicamide eye drops 1% were applied to dilate pupils, and corneas were kept hydrated with 0.3% hypromellose gel (GenTeal Eye Gel, Novartis). Retinal images were recorded using a contact camera for small rodents (Micron IV, Phoenix Research Labs, Pleasanton, CA) under white light illumination to visualize the fundus. GFP-positive cells were imaged using a 469/35 nm exciter filter and a 488 nm long pass barrier filter. Animals were recovered by intraperitoneal injection of atipamezole (2.5 mg/kg).
Genteal eye gel
Manufactured by Novartis
Sourced in Switzerland
GenTeal eye gel is a topical ophthalmic product. It contains hydroxypropyl methylcellulose, which is a lubricating agent that can help maintain moisture in the eyes.
Automatically generated - may contain errors
5 protocols using genteal eye gel
1
Anesthetized Rodent Retinal Imaging
2
In Vivo Fundus Imaging in Mice
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To perform fundus examination, mice were anaesthetized by an intraperitoneal injection of ketamine (80 mg/kg) and xylazine (10 mg/kg). The pupils were then dilated with 0.5% tropicamide drops (Mydriacil; Alcon, Geneva, Switzerland), and corneas were kept moist with GenTeal eye gel (Novartis, Basel, Switzerland). Fundus imaging was performed using a Micron III retinal imaging system (Phoenix Research Laboratories, Pleasanton, CA, USA) with StreamPix 5 software. Brightness and contrast adjustments were uniformly applied to images in Adobe Photoshop. Immediately after in vivo imaging, mice were euthanized for tissue collection.
3
In vivo Intravital Microscopy Imaging
4
Intravital Imaging of EGFR in Tumor Vasculature
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For intravital imaging, mice harboring HSC3/EGFR-GFP cell-derived tumors (150–250 mm3) were anesthetized (Ketamine 100 mg/kg, Xylazine 12.5 mg/kg). Tumors were exposed by cutting the skin surrounding the tumor and holding the tumor steady with metal lifter to minimize motion artefacts caused by heart beat and breathing. To monitor EGF-Rh in tumors, 5 μg of EGF-Rh in 100 μl PBS was injected in the tail vein, and imaging was performed 1 hr after the injection. GenTeal eye gel (Novartis; Basel, Switzerland; refractive index 1.33) was placed on the tumor to interface with the two-photon objective. The eye gel has the same refractive index as water and is readily compatible with water immersion objectives. Time lapse image series were acquired using a Nikon multiphoton microscope (with 25x WI objective 1.15NA) at 1/4 frames per second with excitation at 800 nm and emission through 500–550 nm and 570–620 nm filters for GFP and rhodamine detection, respectively.
5
Two-photon intravital imaging of the thoracic microvasculature
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After administration of an intravascular fluorescent dye and fluorescent mAbs, mice were transported on the heated stage to the microscope stage. The microscope stage was enclosed in a temperature controlled black plexiglass enclosure set at 37 °C, which prevented interference by the ambient light. GenTeal eye gel (Novartis; Basel, Switzerland; refractive index 1.33) was placed on the glass coverslip of the thoracic window device to interface with the two-photon objective. The eye gel has the same refractive index as water and is readily compatible with water immersion objectives. The approximate imaging plane was focused using the fluorescent lamp and eye piece, with fine adjustments made through the Nano-Drive and Prior stage following initiation of scanning in the resonant mode. Movies were acquired using NIS Elements software with the following settings: resonant scan mode with 16x line averaging (1.9 frames per second) and bi-directional scanning, laser power of 5%, and excitation wavelengths of 920 nm and 940 nm for dual color movies and 850 nm for tricolor movies. Acquisition was often conducted using a field of view of 512 µm x 512 µm or 260 µm x 260 µm at 1 or 0.5 µm resolutions, respectively.
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