Samples were prepared for imaging by placing a vacuum grease coated O-ring on microscope cover glass. The sample was then applied to the center of the well and sealed with a coverslip. For each sample, 1 μl of PEG-coated NPs were added to 20 μl human mucus (approximately 2 × 106 particles/sample) in the center of the slide well and stirred with a pipette tip prior to imaging. Samples were then equilibrated for 30 min at room temperature prior to imaging. Slides were imaged using a Zeiss LSM 800 inverted microscope with a 63× water-immersion objective. Multiple 10 s videos were recorded at 33.3 frames per second for each sample. Similar methods were used for measuring influenza A virus diffusion in human mucus and are described in detail in our previously published work.15 (link)
Lsm 800 inverted microscope
Manufactured by Zeiss
Sourced in Germany
The Zeiss LSM 800 is an inverted microscope designed for high-resolution imaging. It features a confocal laser scanning system that allows for optical sectioning and three-dimensional reconstruction of samples. The microscope is equipped with a set of objectives and detection channels to support a variety of imaging techniques.
Automatically generated - may contain errors
5 protocols using lsm 800 inverted microscope
1
Nanoparticle Diffusion in Human Mucus
2
Morphological Analysis of Neuronal Dendritic Spines
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Morphological analysis and spine type classification were performed as previously published (Calfa et al., 2012 (link); Giachero et al., 2015 (link)). Briefly, animals received a challenge injection of saline or cocaine and, 24 h later, animals were deeply anesthetized before being perfused transcardially. Sections of the brain were made with a vibratome. Dendritic portions of NA core cells were stained with saturated solution of lipophilic dye (DiI, Invitrogen Carlsbad, CA, USA) in fish oil (Pozzo-Miller et al., 1999 (link)) by microinjection with a patch pipette and positive pressure application. Z-sections from labeled dendritic segments were collected using a confocal microscope (Carl Zeiss LSM 800 inverted microscope).
3
Confocal Imaging of Live Cells
4
Mitochondrial ROS detection in photodynamic therapy
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2 × 105 cells were seeded in 35 mm glass-bottom dishes and allowed to adhere overnight. On the next day, cells were pretreated with dark HYP (2 μM, without irradiation), or irradiated HYP (2 μM, for 2 min), irradiated HYP (2 μM, for 2 min) +Lip-1 (8 μM). 2 h after irradiation, cells were stained with Mito Tracker Green FM (200 nM, Invitrogen) for 30 min at 37 °C., Images were acquired using a LSM800 inverted microscope (Zeiss) with a 63 × 1.4 NA oil immersion objective. 488 nm and 561 nm laser lines were used to excite the Mito Tracker Green FM and HYP, respectively.
To detect mitochondrial ROS, the PDT treated cells were first stained with Mito Tracker Green FM for 30 min at 37 °C, and then washed twice with PBS before staining with 1.25 μM MitoSOX red (Invitrogen) for another 30 min at 37 °C. Images were acquired using CLSM with 488 nm and 561 nm laser lines to excite Mito Tracker Green FM and MitoSOX, respectively.
To detect mitochondrial ROS, the PDT treated cells were first stained with Mito Tracker Green FM for 30 min at 37 °C, and then washed twice with PBS before staining with 1.25 μM MitoSOX red (Invitrogen) for another 30 min at 37 °C. Images were acquired using CLSM with 488 nm and 561 nm laser lines to excite Mito Tracker Green FM and MitoSOX, respectively.
5
Evaluating Glioblastoma Spheroid Responses
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The GL261-GFP spheroids that reached a diameter of 0.3 mm were carefully transferred on top of the organotypic brain slices using a 200-µL tipped pipette (one spheroid per organotypic brain slice) and incubated at 37 °C and 5% CO 2 . Twenty-four hours after spheroid inoculation, brain organotypic slice bearing GL261-GFP spheroids were treated with 1 μM of DOXC 12 (prediluted in 0.1% of DMSO) and 1 μM of DOXC 12 -LNC CL solution or blank LNC CL , appropriately diluted in the brain-culture medium. The organotypic slices were cultivated at 37 °C and 5% CO 2 for 2 weeks and the drug-containing brain-culture medium was replaced every 2 days. The slices were scanned using a fluorescence LSM800 inverted microscope (Zeiss, Germany) with excitation filters of GFP to evaluate the presence and monitor the size of the spheroids. Data were normalized to time zero and fluorescence intensity was quantified by ImageJ software (Fiji software, v.1.53f51).
In vivo studies: anti-cancer efficacy of DOXC 12 in orthotopic GL261-bearing models
In vivo studies: anti-cancer efficacy of DOXC 12 in orthotopic GL261-bearing models
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