Cultures were imaged using a fluorescent stereoscope (Nikon SMZ1270i) every 48 hours post-culture establishment, and every 24 hours post-electroporation monitor for morphological, structural, or fluorescent protein expression changes within the tissue. The Zeiss LSM 710 confocal microscope was used to image tissue cultures after fixation alone or fixation and staining. Images were analyzed using FIJI version 2.9.0.
Smz1270i
Manufactured by Nikon
Sourced in Japan
The SMZ1270i is a stereo zoom microscope designed for a wide range of laboratory applications. It offers a zoom range of 12.7:1 and a maximum magnification of 288x. The microscope features high-quality optics and a sturdy, ergonomic design.
Automatically generated - may contain errors
Lab products found in correlation
Digital sight ds fi2, by Nikon (5 mentions)
Ms 222, by Merck Group (3 mentions)
Nis elements d analysis software version 3.2, by Nikon (3 mentions)
Lsm 710 confocal microscope, by Zeiss (3 mentions)
Nis elements analysis d software, by Nikon (2 mentions)
Ampicillin, by Merck Group (1 mentions)
Rifampicin, by Merck Group (1 mentions)
Mouse anti kdel, by Santa Cruz Biotechnology (1 mentions)
Bodipy solution, by Thermo Fisher Scientific (1 mentions)
Rhodamine phalloidin, by Thermo Fisher Scientific (1 mentions)
A1 confocal microscope, by Nikon (1 mentions)
Lsm880 fastairy, by Zeiss (1 mentions)
Lsm710 meta big, by Zeiss (1 mentions)
Dragonfly spinning disc microscope, by Oxford Instruments (1 mentions)
Axio observer 7 inverted microscope, by Zeiss (1 mentions)
8 protocols using smz1270i
1
Imaging and Visualization of Cell Cultures
2
Eel Larval Development and Gene Expression
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Larval development (biometry), mortality, and gene expression of selected genes, corresponding to specific molecular mechanisms were followed from hatch and throughout the endogenous feeding stage (2, 4, 6, 8, 10, and 12 dph). All endogenously feeding larvae of European eel were anesthetized using tricaine methanesulfonate (MS-222; Sigma-Aldrich, Missouri, USA) prior to digital imaging and euthanized post-sampling by using an MS-222 overdose [13 (link)]. All images were taken using a digital camera (Digital Sight DS-Fi2, Nikon Corporation, Japan) attached to a zoom stereomicroscope (SMZ1270i, Nikon Corporation, Japan), while NIS-Elements D analysis software (Version 3.2) was used to analyze the larval images (Nikon Corporation, Japan).
3
Quantifying Teleost Embryo Development
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The weight of stripped eggs was recorded prior to fertilization, while ~ 30 min post-fertilization, the amount of buoyant eggs (%) was determined in a 25-mL volumetric column (Tomkiewicz 2012 ).
At 4 hpf, a sample of eggs (n ~ 100 eggs) was obtained and photographed using a stereomicroscope (SMZ1270i, Nikon Corporation, Japan) with a mounted camera (Digital Sight DS-Fi2, Nikon Corporation, Japan). Subsequently, digital images were analysed applying NIS-Elements-D analysis software (Version 3.2, Nikon Corporation, Japan). Using the 4-cell stage as criterion (Sørensen et al. 2016b ), fertilization success was calculated as the percentage of fertilized eggs divided by the total number of eggs imaged. In order to estimate the embryonic survival at 24 and 48 hpf, the number of embryos was calculated in 3 × 15 ml water samples collected from the incubators. For the estimation of hatching success, subsamples of ~ 100 embryos were collected from the incubators at 48 hpf and inserted into 200-mL sterile tissue culture flasks (VWR, Denmark) filled with culture water enriched with rifampicin and ampicillin (each 50 mg L−1, Sigma-Aldrich, Missouri, USA) (Sørensen et al. 2014 ). Approximately 12 h after hatching, the number of larvae and unhatched embryos was recorded to assess hatching success (%).
At 4 hpf, a sample of eggs (n ~ 100 eggs) was obtained and photographed using a stereomicroscope (SMZ1270i, Nikon Corporation, Japan) with a mounted camera (Digital Sight DS-Fi2, Nikon Corporation, Japan). Subsequently, digital images were analysed applying NIS-Elements-D analysis software (Version 3.2, Nikon Corporation, Japan). Using the 4-cell stage as criterion (Sørensen et al. 2016b ), fertilization success was calculated as the percentage of fertilized eggs divided by the total number of eggs imaged. In order to estimate the embryonic survival at 24 and 48 hpf, the number of embryos was calculated in 3 × 15 ml water samples collected from the incubators. For the estimation of hatching success, subsamples of ~ 100 embryos were collected from the incubators at 48 hpf and inserted into 200-mL sterile tissue culture flasks (VWR, Denmark) filled with culture water enriched with rifampicin and ampicillin (each 50 mg L−1, Sigma-Aldrich, Missouri, USA) (Sørensen et al. 2014 ). Approximately 12 h after hatching, the number of larvae and unhatched embryos was recorded to assess hatching success (%).
4
Larval Biometry and Mortality Dynamics
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From 10 dph and onwards, dead larvae were counted and removed daily from all experimental units (n = 9). Larval cumulative mortality was calculated as a percentage from 10 until 28 dph. Larvae sampled throughout the experimental period were not considered for further calculation. Subsamples (n = 3) of 10 larvae each were collected at hatch (0 dph) and larvae photographed for subsequent measurement of initial larval biometry. Thereafter, pools of 10 larvae per replicated tank (n = 3) and diet (n = 3) were sampled and photographed at selected developmental stages, including the beginning of the first-feeding window, but before introduction of the experimental diets (9 dph), the middle of the first-feeding window (15 dph), the end of the first-feeding window (22 dph) and beyond the first-feeding stage (28 dph), passing the point of no return. All images were taken using a digital camera (Digital Sight DS-Fi2, Nikon Corporation, Japan) attached to a zoom stereomicroscope (SMZ1270i, Nikon Corporation, Japan). NIS-Elements D analysis software (Nikon Corporation, Japan, Version 3.2) was used to assess larval biometrics from the images, such as standard length and body area, while feeding incidence and gut fullness at 15 and 22 dph was calculated as described in [21 (link)].
5
Imaging Ovary and Fat Body Structures
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Adult ovaries and larval/adult fat body were dissected in PBS and fixed in 4% PFA for 15 min at RT. Samples were washed in PBS+0.1% detergent (Triton X-100 for ovary, Tween 20 for fat body) twice and incubated overnight at 4°C in primary antibody solution. Secondary antibodies along with DAPI (300nM final concentration) were incubated for 2 h at RT in the dark. Mouse anti-KDEL (Santa Cruz Biotech) was used at 1:100. Phalloidin-Rhodamine (Life Technologies) was used at 1:500. For yolk granule visualization, ovaries were fixed, washed, and mounted without DAPI. Yolk granules were visualized by auto-fluorescence upon excitation by 405nm laser.74 (link) For neutral lipid staining in the adult fat body, samples were fixed and washed, followed by incubation in BODIPY solution (Thermo Scientific) for 20 min at a final concentration of 2 μg/mL. Confocal images were captured using a Nikon A1 confocal microscope through the Center for Biological Imaging at the University of Pittsburgh. Egg retention representative images were captured using a Nikon SMZ1270I with a ring light and DS-Ri2 camera attachment.
6
Zebrafish Embryo Microscopy Techniques
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Embryos were mounted in 35 mm glass bottom dishes using 0.7% low melting point agarose. Confocal z-stacks were acquired on a LSM 710 Meta BiG (Zeiss) and a LSM 880 FastAiry (Zeiss) using a LD C-Apochromat W/40×1.1 NA objective. Timelapse imaging was performed on a Dragonfly Spinning Disc microscope (Andor) using Apo λ LWD W/40×1.15 NA. Bright-field images of the whole-mount zebrafish embryos were taken on a Nikon SMZ1270i with Tucsen Michrome 6 camera. Movies of blood flow (Movies 4 -7 ) were recorded with a Prime BSI Express camera at 100 frames per second and captured using a EC-Plan Neofluar 20×0.8 NA objective on an Axio Observer 7 inverted microscope (Zeiss).
7
Larval Development Biometry in Fish
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In order to follow larval development (biometry) from hatch until the feeding stage, larvae were sampled at 0, 6, and 12 dph. From each replicate (n = 3), family (n = 3), and treatment (n = 4) ~10 larvae were randomly sampled and imaged using a digital camera (Digital Sight DS-Fi2, Nikon Corporation, Japan) attached to a zoom stereomicroscope (SMZ1270i, Nikon Corporation, Japan). All larvae were anesthetized using tricaine methanesulfonate (MS-222; Sigma-Aldrich, Missouri, USA) prior to digital imaging. Subsequently, NIS-Elements-D analysis software (Version 3.2) was used to analyze the larval images (Nikon Corporation, Japan), where total body area (BA) and oil-drop area (ODA) were measured for each larva. Larval growth [(BA (day12) -BA (day0) ) / Age (12 days) ] and oil-drop utilization [(ODA (day0) -ODA (day12) ) / Age (12 days) ] rate were measured from the change in body and oil-drop area, respectively. Growth efficiency was then measured by dividing the increase in body area by the corresponding decrease in oil-drop area (Politis et al., 2014 b) . Moreover, the severity of pericardial edema was calculated using the ratio between edema/neurocranium heights (Okamura et al., 2009) (link).
8
Larval Biometry and Oil-Drop Utilization
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Larval development (biometry) was followed from hatch and throughout the endogenous feeding stage (0, 6, and 12 dph), where ~10 larvae from each replicate (n = 3), family (n = 3), and treatment (n =4) were randomly sampled and digitally imaged using a digital camera (Digital Sight DS-Fi2, Nikon Corporation, Japan) attached to a zoom stereomicroscope (SMZ1270i, Nikon Corporation, Japan). All endogenously feeding larvae were anesthetized using tricaine methanesulfonate (MS-222; Sigma-Aldrich, Missouri, USA) prior to digital imaging. NIS-Elements-D analysis software (Version 3.2) was used to analyze the larval images (Nikon Corporation, Japan), where total body and oil-drop area were measured for each larva. Larval growth and oil-drop utilization rate were measured from the change in body and oil-drop area, respectively. Growth efficiency was then measured by dividing the increase in body area by the corresponding decrease in oil-drop area (according to Politis et al 2014) (link). Moreover, the severity of pericardial edema was calculated using the ratio between edema/neurocranium heights according to Okamura et al 2009.
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