Mice were anesthetized [xylazine hydrochloride (10 mg/kg) and ketamine hydrochloride (200 mg/kg)], and the body temperature was maintained at 37°C using a heating pad (CU-201, Live Cell Instrument). The right jugular vein was cannulated to administer fluorescent dyes and additional anesthetic. The trachea of the mouse was exposed, and a small catheter was threaded into the trachea. The catheter was then attached to a small rodent ventilator (Harvard Apparatus). The mouse was placed on its right lateral decubitus position. A small surgical incision was made, and the intercostal muscles between ribs 4 and 5 were gently teased apart forming a ~1.5-cm opening. Intercostal lung window was then carefully placed between ribs 4 and 5. The lung was stabilized with a suction of ~20 mmHg. Images were acquired with a Leica Sp8 upright microscope equipped with a resonance scanner.
Sp8 upright microscope
Manufactured by Leica
The Leica SP8 upright microscope is a versatile research-grade instrument designed for high-performance imaging. It features advanced confocal technology, providing users with detailed, high-resolution images of a wide range of samples. The SP8 microscope is capable of performing a variety of imaging techniques, making it a valuable tool for various scientific applications.
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Lab products found in correlation
4 protocols using sp8 upright microscope
1
In Vivo Imaging of Mouse Lung Microvasculature
2
Confocal Microscopy of Immuno-labeled Tissue
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Immuno-labeled PCX sections were viewed on a Leica SP8 upright microscope equipped with HC Plan-Apochromat 40× (oil numerical aperture, NA: 1.3) or 100× (oil DIC; NA 1.4) objectives and attached to a spectral confocal laser system with Argon/2 (458, 488, and 514 nm), 561 nm Diode, and HeNe 633 nm. The tissue was scanned with 488-nm and 561-nm laser lines to detect the corresponding Alexa fluorophores. High-resolution images (1024 × 1024 pixels) of optical sections (z-slices) were captured using sequential line (average of 3) scanning with 0.3-μm z-steps.
3
Microscopic Imaging of Fixed Animals
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Adult animals were harvested from a plate; they were washed off in M9 buffer. After three additional washes in M9 buffer, the pelleted animals were fixed in 0.5 ml of precooled methanol at −20°C for 10 min. The pellet was washed three times in Tris-buffered saline containing 0.1% Tween 20 (TBST) and then incubated for 10 min in a 4′,6′-diamidino-2-phenylindole (DAPI) solution (0.5 mg/ml in TBST). The pellet of animals was washed three times in TBST. Five microliters of the pelleted animals was pipetted directly onto a Cel-line diagnostic microscope slide (Thermo Scientific) and imaged using a Leica SP8 upright microscope.
4
Two-Photon Microscopy for Calcium Dynamics
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Two-photon microscopy images were collected using a customized dual Laser (InSight & Mai Tai both Spectra-Physics) Leica SP8 Upright Microscope equipped with a 25x and 0.95 numerical aperture water-immersion objective. Three channels were acquired simultaneous using a resonant scanner and lasers tuned to 910 and 985nm. Fluorescence emission was guided directly to external hybrid photodetectors (Leica). For signal separation, we used three separate dichroic beam splitters (without bandpass filters) FF640-FDi01, FF562-FDi03 and FF506-FDi03 (Semrock). The mirrors were arranged in dendritic fashion where settings generated four channels: approximately 390–506nm, 506–562nm, 562–640nm and 640–680nm (not recorded). For analysis, the cell surfaces were defined and the mean fluorescence intensities (green/GCamp6f and red/TdTomato) of the cell region were manually measured in ImageJ for all flashing cells within 150μm of the wound edge. The calcium spike amplitude was determined as (green/redpeak)/(green/redbaseline). The spike duration was defined as the time during which calcium amplitude >1.1.
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