For immunofluorescence labeling, the deparaffinated section was stained with anti-perilipin (1:100, Abcam, Cambridge, MA, USA), anti-GFP (1:200, Abcam), and 4′,6-Diamidino-2-Phenylindole (DAPI, 0.5 µg/mL, Invitrogen, Carlsbad, CA, USA). A confocal microscope (Leica SP8 MP, Wetzlar, Germany) was used to detect fluorescence images. Quantification of fluorescence signal was determined by ImageJ software (USA).
Sp8 mp
Manufactured by Leica camera
Sourced in Germany
The SP8-MP is a multi-photon confocal microscope from Leica. It is designed for high-resolution imaging of thick samples and living specimens. The system utilizes state-of-the-art laser technology and advanced optics to provide exceptional image quality and performance.
Automatically generated - may contain errors
Lab products found in correlation
Chambered coverglasses, by Thermo Fisher Scientific (2 mentions)
Lsm 880, by Leica camera (2 mentions)
Anti perilipin, by Abcam (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Anti gfp, by Abcam (1 mentions)
Qtracker 655, by Thermo Fisher Scientific (1 mentions)
Hyd detector, by Leica camera (1 mentions)
Xylenol, by Merck Group (1 mentions)
Oxytetracycline hcl, by Merck Group (1 mentions)
Alizarin red s, by Merck Group (1 mentions)
8 protocols using sp8 mp
1
Immunofluorescence Labeling of Adipocytes
2
Confocal microscopy imaging protocols
3
Intravital Imaging of Murine Tibial Bone Marrow
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Hlf-tdTomato KI adult and neonatal mice were anesthetized with isoflurane using an inhalational anesthesia system34 (link)–36 (link). Anesthesia was maintained using 1.0–1.5% isoflurane at a flow rate of ~0.2 L/min air. Anesthetized mice were placed on heating pads and monitored to maintain body temperature. The skin around the tibia was cut and the tibia was fixed to reduce the artifacts caused by the heartbeat and breathing to image the tibial bone marrow. A coverslip was placed on the exposed tibia. Intravenous injection of Qtracker 655 (Invitrogen) was conducted by retro-orbital injection (in adult mice) or superficial temporal vein injection using a pulled glass capillary (in neonatal mice) immediately prior to imaging. Intravital imaging of the tibial bone marrow was performed using a customized multiphoton laser-scanning upright microscope (Leica, SP8MP) with a 25× water immersion objective having a numerical aperture of 0.95 (Leica) and fiber oscillators that deliver pulses at 1070 nm (Coherent, Fidelity-2) and 920 nm (Spark lasers, Alcor-920). Fluorescence was collected by the 25× objective and directed to two HyD detectors (Leica) and one photomultiplier tube. Three-dimensional images of the bone marrow with a field of view of ~600 μm × 600 μm (512 × 512 pixels) were acquired every 5 μm.
4
Cardiomyocyte Energetics: Mitochondrial and Cytosolic
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We isolated mature cardiomyocytes from the indicated genotype mice using a Langendorff perfusion system (Radnoti, USA). To investigate the effect of empagliflozin, cariporide, and ranolazine on cardiomyocyte energetics in mitochondria or cytosol, we studied the mitochondrial or cytosolic ATP levels of cardiomyocytes with/without empagliflozin (10, 100, 300, 1000 nM), cariporide (10 μM), ranolazine (30 μM) or MA-5 (10 μM). To investigate the effect of empagliflozin on the mitochondrial membrane potential of cardiomyocyte, we used the JC-1 with empagliflozin (300 nM). Two-photon microscopy (Leica SP8MP) was used to observe fluorescence.
5
Visualizing Bone Remodeling Dynamics
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After 8 weeks of healing, the dynamics of the newly formed bones were observed by injecting them with fluorescent bone marker labels. At 2 weeks, alizarin red S (20 mg/kg body weight), oxytetracycline HCl (20 mg/kg body weight), and xylenol (20 mg/kg body weight) (Sigma-Aldrich) were administered via intraperitoneal injection. All dyes were prepared with saline immediately before use. To visualize remodeled bone, an intravital multiphoton microscope (SP8-MP; Leica, Wetzlar, Germany) with a water immersion lens (25×, 0.9 NA) was used. The fluorophores were excited using the InSight DS Plus laser system (Spectra-Physics, Santa Clara, USA) at the following wavelengths: xylenol (excitation = 800 nm, emission = 375-570 nm), oxytetracycline (excitation = 1040 nm, emission = 365-490 nm), and alizarin (excitation = 1180 nm, emission = 538-580 nm). Alizarin -, oxytetracycline-, and xylenol-positive areas were quantified using the ImageJ software. All values are expressed as mean ± SD (n = 4).
6
Confocal microscopy imaging protocols
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Confocal laser-scanning microscopy images were obtained using either a Zeiss LSM 880 (with Zen 2.1 SP3 Black edition), Leica SP8 (with LasX 3.1.5.16308) or Leica SP8-MP (with LasX 3.5.6.21594) microscopes. For green and red fluorophores, the following excitation and detection windows were used: mVENUS/GFP/FY/FDA 488 nm, 500-530 nm; mCITRINE 496 nm, 505-530 nm; PI 520 nm, 590-650 nm; Calcofluor White 405nm, 430-485 nm; Basic Fuchsin/Nile Red 561nm, 600-630 nm. For multiphoton microscopy the following excitation and detection settings were used: mVENUS/GFP/FY/Calcofluor White 960 nm, 435-485 nm (Calcofluor White) and 500-550 nm (mVENUS/GFP/FY). Methods for imaging the CS lignin and PI penetration were previously described25 (link),28 (link). For visualization of FDA transport, chambered cover glasses (Thermo Scientific), were used where the roots were covered with a slice of agar and time lapses were made right after the application of FDA.
7
In vivo skull bone imaging
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In vivo BM imaging of the skull bone in live animals was described previously [54 (link)]. Fifty μl of Cy5-conjugated dextran (8 mg/ml, Yuanye, Shanghai, China) was injected via the tail vein to stain the blood vessel. By exposing the skull bone, fluorescence images were acquired by using a two-photon confocal microscope (Leica SP8 MP).
8
Intravital Microscopy for Bone Regeneration
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To image bone regeneration, we used two-photon fluorescence microscopy (TPM) with an intravital multiphoton microscope (SP8-MP; Leica, Wetzla) at the Korea Basic Science Institute (KBSI, Gwangju, Korea). Bone samples were stored in PBS for TPM imaging. The fluorophores were irradiated using the InSight DS Plus laser system (Spectra-Physics, Santa Clara, CA, USA) at an excitation wavelength of 800 nm (xylenol), 1040 nm (oxytetracycline), and 1180 nm (alizarin). Emissions were collected at 365-490 nm (oxytetracycline), 375-570 nm (xylenol), and 538-580 nm (alizarin).
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