Zeiss LSM780 and Zeiss LSM800 GaAsP single‐point laser‐scanning microscope were used for confocal image acquisition, where bottom focal planes were focused on to capture basal cortical waves.
Lsm800 gaasp
Manufactured by Zeiss
The LSM800 GaAsP is a laser scanning microscope system designed by Zeiss. It is equipped with GaAsP (Gallium Arsenide Phosphide) detectors, which provide high sensitivity and low noise performance. The core function of the LSM800 GaAsP is to enable high-resolution imaging and analysis of samples.
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Lab products found in correlation
Lsm780 fcs, by Zeiss (2 mentions)
Lsm 780, by Zeiss (1 mentions)
Chambered coverglass, by Grace Bio-Labs (1 mentions)
Lsm 510 meta, by Zeiss (1 mentions)
Lsm 880 airyscan, by Zeiss (1 mentions)
Zen imaging software, by Zeiss (1 mentions)
Axio imager, by Yokogawa (1 mentions)
Imaris software v9, by Oxford Instruments (1 mentions)
Cm3050s cryostat, by Leica (1 mentions)
P histone h3, by Cell Signaling Technology (1 mentions)
Alexa 488 anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Alexa 568 anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Alexa 568 anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Tom20, by Proteintech (1 mentions)
7 protocols using lsm800 gaasp
1
Basal Cortical Wave Imaging
2
Fluorescence Recovery After Photobleaching
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20 µL condensation reactions (prepared as described above) were added to a chambered coverglass (Grace BioLabs) and imaged using a Zeiss LSM 800 GaAsp. Bleaching was performed using 100% laser power in the 488, 546, or 647 channels. Regions slightly larger than the condensates (radius ≈ 3 µM) were photobleached. A single confocal plane was imaged using a 63x objective every 3 s during a recovery phase of 300 s.
FRAP analysis was performed as described in Putnam et al. (2019) (link). Briefly, fluorescence recovery was corrected for background and normalized to the initial granule intensity using the equation: nI = (I-Ibkg)/(Ii-Ibkgi), where nI is the background corrected and normalized fluorescence intensity, I is the intensity of the FRAPed granule, Ibkg is the fluorescence intensity outside of the condensate, Ii is the initial intensity before bleaching, and Ibkgi is the initial background intensity. Recovery rates were determined by fitting individual traces to a first order equation nI = (Arec·(1-e-k t ), where Arec is the fluorescence recovery amplitude and k is the rate of fluorescence recovery. For RNAs where fluorescence recovery was in the linear range for the entire time course, initial recovery rates were calculated by fitting to a linear equation nI = kt, where k is the initial rate of fluorescence recovery (Figure 5D , Figure 5—figure supplement 1B ).
FRAP analysis was performed as described in Putnam et al. (2019) (link). Briefly, fluorescence recovery was corrected for background and normalized to the initial granule intensity using the equation: nI = (I-Ibkg)/(Ii-Ibkgi), where nI is the background corrected and normalized fluorescence intensity, I is the intensity of the FRAPed granule, Ibkg is the fluorescence intensity outside of the condensate, Ii is the initial intensity before bleaching, and Ibkgi is the initial background intensity. Recovery rates were determined by fitting individual traces to a first order equation nI = (Arec·(1-e-
3
Confocal Microscopy Imaging Protocol
4
Fluorescence Microscopy Imaging of Embryos
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Fluorescence confocal microscopy was performed using a Zeiss Axio Imager with a Yokogawa spinning-disc confocal scanner. Embryo images were taken using Slidebook v6.0 software (Intelligent Imaging Innovations) using a 63x objective. Embryos were staged by DAPI-stained nuclei in optical Z-sections and multiple Z-sections were taken to include germ cells. For In vitro condensation reactions, images are single planes taken using a 40x objective unless otherwise indicated. For fluorescence super-resolution microscopy, images were acquired using ZEISS LSM 880-AiryScan (Carl Zeiss) equipped with a 63X objective. Images were processed using ZEN imaging software (Carl Zeiss). Equally normalized images were exported via either Slidebook v6.0 or ZEN, and contrasts of images were equally adjusted between control and experimental sets. For in vitro fluorescence recovery after photobleaching experiments, images were acquired using Zeiss LSM 800 GaAsp. Images are single confocal planes imaged using a 63x objective every 3 s during a recovery phase of 300 s. All image analyses were conducted using the Fiji image-processing package (http://fiji.sc/Fiji ).
5
Quantification of Nerve Fibers and Vasculature
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Images for quantification were obtained with confocal microscopy (Zeiss LSM780 FCS, or LSM800 GaAsP, Carl Zeiss Microscopy GmbH). TUBB3+ nerve fibers, CD31+, Emcn+ and CD146+ blood vessels were quantified using three-dimensional volumetric analysis of Imaris software v9.3 (Oxford Instruments, Belfast, UK) using eight serial fields per sample within the injured tissue, which included the distal tenotomy site and immediately surrounding tissues. For quantification of angiogenic factors within the DRG, cross sections of the whole ganglia were used with Leica DM6 B (Wetzlar, Germany) imaging and ImageJ software.
6
Visualizing Gli1-Expressing Cells in Mouse Brain
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Gli1LacZ/+ mice were transcardially perfused with PBS and 4% PFA. The isolated brains were fixed in 4% PFA at 4°C overnight, transferred into 30% sucrose at 4°C for 48 h, and embedded into optimal cutting temperature compound. The 40 µm sagittal cryosections were collected using Leica Cryostat CM 3050S and mounted on the glass slides, which were dried with natural air at room temperature. The fixation buffer (2% paraformaldehyde, 0.02% glutaraldehyde, 2 mM MgCl2 in PBS) was used to fix slides for 10 min at room temperature, followed by a 10 min PBS wash and another 10 min wash using wash buffer (5 mM EGTA, 0.01% Deoxycholate, 0.02% NP40, 2 mM MgCl2 in 0.1 M phosphate buffer). The slides were then incubating with X-gal staining solution [5 mM K3Fe(CN)6, 5 mM K4Fe(CN)6, 5 mM EGTA, 0.01% Deoxycholate, 0.02% NP40, 2 mM MgC12, 1 mg/ml X-gal] at 37°C in the dark overnight, followed by a wash in PBS for 10 min and a wash in distilled water for 5 min. X-gal stained slides (Fig. 2A and Supplementary Fig. 2) were co-stained with anti-Calbindin antibodies for confocal imaging (Zeiss LSM800 GaAsP, MicFac).
7
Immunofluorescence Imaging of Mitochondrial Dynamics
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MEFs were fixed in pre-warmed 4% paraformaldehyde in PBS at room temperature for 20 minutes on an 8-well chambered cover glass.13 (link),62 (link),71 (link) They were then permeabilized using PBS containing 0.1% Triton X-100 at room temperature for 8 minutes. Following permeabilization, the cells were blocked in 0.5% BSA/PBS at room temperature for 30 minutes. Overnight incubation with primary antibodies in 0.5% BSA/PBS was conducted at 4°C, which was followed by a 1-hour incubation with fluorescently labeled secondary antibodies in PBS at room temperature. The primary antibodies used included pDrp1 S579 (1:200 dilution), pHistone H3 (1:200 dilution), Drp1 (Cell Signaling Technology, 8570; 1:200 dilution) for Figure 4 E, Drp1 (BD Biosciences, 611113; 1:200 dilution) for Figure 5 C, and TOM20 (Proteintech, 11802-1-AP; 1:300 dilution). The secondary antibodies (Invitrogen), used at a 1:400 dilution, were Alexa 488 anti-rabbit IgG, Alexa 568 anti-mouse IgG, Alexa 488 anti-mouse IgG (A21202), and Alexa 568 anti-Rabbit IgG (A10042). Imaging was performed using a Zeiss LSM800 GaAsP laser scanning confocal microscope with a 63× objective. NIH ImageJ software was used for image analysis.
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