Fixed sperm and singularized germ cells or HEK293F cells were settled on poly-l -lysine-coated slides overnight and permeabilized with phosphate buffered saline with 1% Triton X-100 (PBST) for 15 min. Slides were washed with PBS and then blocked with 10% horse serum for 30 min before proceeding with antibody staining. Slides were stained with rabbit and/or mouse primary antibodies at a 1:200 dilution in PBS for 2 h, sequentially washed with three changes of PBS for 5 min, and then incubated with anti-mouse and/or anti-rabbit Alexa-conjugated secondary antibodies for 1 h. Peanut agglutinin (PNA) and/or phalloidin were used at a 1:400 dilution for 1 h, coincident with secondary antibodies when colabeling. DAPI was used to counter-stain nuclei at a dilution of 1:500 for 3 min. Slides were then mounted with Aqua-Polymount (#18606, Polysciences, Warrington, PA, USA) and imaged at 63× using a Leica Dmi8 S Platform (LAS X software V5.0.2) inverted microscope system for widefield microscopy or a Zeiss LSM 780 multiphoton microscope (ZEN Blue software V3.4) for confocal microscopy. Images of germ cells and HEK293F cells were assessed for localization patterns relative to cell type and (for germ cells) developmental step.
Lsm 780 multiphoton microscope
Manufactured by Zeiss
Sourced in Germany
The LSM 780 multiphoton microscope is a advanced imaging system designed for high-resolution, 3D imaging of live biological samples. It utilizes multiphoton excitation technology to enable deep tissue imaging with minimal phototoxicity. The system features a high-sensitivity detection system and a flexible optical configuration to accommodate a wide range of sample types and applications.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 710 laser confocal anisotropy, by Zeiss (2 mentions)
Fluoromount g, by Southern Biotech (2 mentions)
Dmi8 s platform, by Leica camera (1 mentions)
Aqua poly mount, by Polysciences (1 mentions)
Glass bottom cell culture dish, by Greiner (1 mentions)
Isofluorane, by Abbott (1 mentions)
Air objective, by Zeiss (1 mentions)
Zen software, by Zeiss (1 mentions)
8 protocols using lsm 780 multiphoton microscope
1
Immunolabeling of Germ and Somatic Cells
2
Microscopy Analysis of GUV Lipid Composition
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For the microscopy experiments, the GUVs were placed into a glass bottom cell culture dish (Greiner bio-one) and incubated with 400 nM Atto633-RICK:20 nM siRNA-Cy3b. Confocal images of GUVs were immediately obtained with an inverted LSM780 multi-photon microscope (Zeiss). The obtained confocal images were projected and treated with the software ImageJ.
3
Multiphoton Microscopy of Anaesthetized Mice
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Mice were anaethetised and surgery performed similar to previously described [23] except that anaethesia was maintained by inhalation of 4% isofluorane (Abbott laboratories, UK). Images were acquired on an inverted LSM 780 multiphoton microscope (Carl Zeiss Microimaging), maintained at 36 °C by a blacked-out environmental chamber (Solent Scientific, UK). Images were acquired with a 40x 1.1 water immersion objective and fluorescence excitation provided by a Chameleon XR Ti:sapphire laser (Coherent) tuned to 870 nm.
4
Second Harmonic Generation Imaging of Tumors
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Unfixed, hydrated control (n = 3) and PSC-depleted (n = 3) tumors embedded in OCT compound were sectioned at 15 μm thickness. The SHG images were acquired with a Zeiss LSM 780 multiphoton microscope mounted on a Zeiss Observer Z1 inverted microscope stand. Tissues were imaged with a Coherent Chameleon Vision II ultrafast pulsed infrared laser (680–1080 nm) tuned to 800 nm. A Zeiss 20× air objective (NA = 0.8) focused the beam onto the sample, and SHG imaging was detected from the back-scattered signal with a Gallium arsenide phosphide non-descanned detector. For image analysis, three representative fields of view were analyzed per sample. A minimal threshold in the SHG signal was set and maintained across all images with ImageJ (NIH) and was used to obtain the percent area and normalized intensity.
5
Analyzing ERK1/2 Activation in MEFs
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WT-MEFs were serum-starved with 0.2% FBS containing DMEM (low serum DMEM) for 12 hours, detached, held in suspension for 30mins in the absence or presence of AZD1152, and re-plated on fibronectin (2µg/ml) for 15mins. Re-adherent cells were fixed with 3.5% paraformaldehyde after 15minutes of re-plating. Cells were permeabilized with PBS containing 5% BSA and 0.05% Triton-X-100 for 15 minutes and blocked with 5% BSA for 1 hour at room temperature followed by incubation with 1:200 rabbit anti-phospho-p44/p42 ERK1/2 (Thr202/Tyr204) antibody in 5% BSA for 3 hours. Cells were finally stained with 1:1000 diluted secondary antibodies (anti-mouse Alexa-568) and 1:500 diluted phalloidin-Alexa 488
for 1 hour at room temperature. All incubations were done in a humidified chamber. Washes were done with 1X PBS at room temperature. Stained and washed coverslips were mounted with Fluoromount-G (Southern Biotech) and imaged using a Zeiss LSM 710 laser confocal-Anisotropy or LSM780 multiphoton microscope with a 63x objective.
for 1 hour at room temperature. All incubations were done in a humidified chamber. Washes were done with 1X PBS at room temperature. Stained and washed coverslips were mounted with Fluoromount-G (Southern Biotech) and imaged using a Zeiss LSM 710 laser confocal-Anisotropy or LSM780 multiphoton microscope with a 63x objective.
6
Analyzing ERK1/2 Activation in MEFs
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WT-MEFs were serum-starved with 0.2% FBS containing DMEM (low serum DMEM) for 12 hours, detached, held in suspension for 30mins in the absence or presence of AZD1152, and re-plated on fibronectin (2µg/ml) for 15mins. Re-adherent cells were fixed with 3.5% paraformaldehyde after 15minutes of re-plating. Cells were permeabilized with PBS containing 5% BSA and 0.05% Triton-X-100 for 15 minutes and blocked with 5% BSA for 1 hour at room temperature followed by incubation with 1:200 rabbit anti-phospho-p44/p42 ERK1/2 (Thr202/Tyr204) antibody in 5% BSA for 3 hours. Cells were finally stained with 1:1000 diluted secondary antibodies (anti-mouse Alexa-568) and 1:500 diluted phalloidin-Alexa 488
for 1 hour at room temperature. All incubations were done in a humidified chamber. Washes were done with 1X PBS at room temperature. Stained and washed coverslips were mounted with Fluoromount-G (Southern Biotech) and imaged using a Zeiss LSM 710 laser confocal-Anisotropy or LSM780 multiphoton microscope with a 63x objective.
for 1 hour at room temperature. All incubations were done in a humidified chamber. Washes were done with 1X PBS at room temperature. Stained and washed coverslips were mounted with Fluoromount-G (Southern Biotech) and imaged using a Zeiss LSM 710 laser confocal-Anisotropy or LSM780 multiphoton microscope with a 63x objective.
7
Detailed Microscopic Analysis of Forv Infection
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The inoculated in vitro plants were examined for Forv infection and colonization at 2, 4, 7, and 9 dpi in at least two independent experiments. Infected roots were carefully taken out of the tubes and thin sections (90 μm) were obtained using a vibrating blade microtome as previously mentioned. These sections were placed in 10 mM phosphate buffer saline (PBS) and then stained with DAPI 300 nM (4′, 6-diamidino-2-phenylindole) for 5 min in the dark. DAPI, classical DNA dye, also stains polyphosphates and emits a yellow fluorescence. The stained cells were then washed twice in PBS. The sections were mounted on a glass slide and observed using a Zeiss LSM780 multiphoton microscope (Zeiss, Germany), equipped with a Chameleon Ultra II laser (Coherent, CA, USA). With the multiphoton microscope, the optimal excitation wavelength for DAPI is 720 nm and the filter blocks, with differential spectral properties, were set to those of DAPI (415–480 and 550–610 nm) and chlorophyll (660–700 nm). Image acquisition was performed using Zen software (Zeiss, Germany). The acquired image channels were merged and processed using Image J 1.47v software.
8
SHG Imaging of Collagen Fibrils
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Fibrillary collagen in non-fixed cell-embedded collagen gels was imaged using the SHG technique (Chen et al., 2012 (link)) with a Zeiss LSM780 multiphoton microscope (Carl Zeiss Microscopy; Jena, Germany) fitted with a short pulse laser.
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