Cells grown on coverslips were fixed using 4% PFA for 20 minutes at room temperature (RT). Cells were permeabilized and blocked using a buffer containing 5% Horse/Donkey/Goat serum, 3% BSA and 0.3% TritonX-100 for 30 minutes at RT. Primary antibodies diluted in antibody diluent (1% Horse/Donkey/Goat serum + 3% BSA + 0.1% TritonX-100) were incubated overnight at 4°C. Secondary antibodies were incubated for 1h at RT. Cells were washed three times with PBS + 0.1%Tween-20. Hoechst (4 μg/mL) was used to visualize nuclei. Coverslips were mounted on slides using Prolong Gold mounting medium (Life Technologies). Images were acquired using confocal microscope Radiance 2100 (Zeiss, Germany) equipped with an upright microscope (Eclipse E800; Nikon, Japan). The Supplementary Table 5 brings a detailed description of the antibodies used in this study.
Radiance 2100
Manufactured by Zeiss
Sourced in Germany
The Radiance 2100 is a high-performance microscope imaging system designed for advanced scientific and research applications. It features a sophisticated optical system that delivers exceptional image quality and resolution. The Radiance 2100 is capable of capturing detailed, high-resolution images and videos for a wide range of sample types and applications.
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Lab products found in correlation
9 protocols using radiance 2100
1
Immunofluorescence Staining of Cultured Cells
2
Visualizing Motor Axons in Transgenic Zebrafish
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To visualize motor axons in GFP transgenic animals, Tg(mnx1:GFP) zebrafish were fixed with 4% paraformaldehyde in PBS overnight at 4°C, washed with PBST for 10 min, three times and mounted on slides for observation using a Zeiss Radiance 2100 laser scanning system together with Laser-Sharp and LSM imaging software (Carl Zeiss MicroImaging, Inc.). Ten motor axons from each side of the embryo were scored (total of 20 per embryo) at 28 h post-fertilization and used to classify the embryo as one truncation, two truncations, three truncations or no defect. Three sets of injections of embryos spawned the same day from different parents were performed per condition and 200 embryos (4000 motor axons) scored in each experiment. Statistical analysis was carried out using Prism 6 (GraphPad) software. The distribution of larval classifications was analyzed by comparing median values of each group with a two-tailed student's t test. Data are represented as mean and standard error of the mean (SEM) from independent experiments and P values are indicated as follows: *P < 0.05; **P < 0.01; ***P < 0.001.
3
Immunofluorescence Staining of hCLE
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Cells were fixed with 3.7% formalin in PBS during 20 min at room temperature and permeabilised for 5 min with 0.5% Triton X-100 in PBS. The preparations were blocked with 1% bovine serum albumin (BSA) in PBS for 30 min and then incubated for 45 min with the following primary antibodies: a rabbit polyclonal antibody against hCLE (1∶1000) [1] (link), a goat polyclonal antibody against DDX1 (1/50), and a goat polyclonal antibody against HSPC117 (1/500). After washing with PBS, the preparations were incubated with the corresponding secondary antibodies and DAPI using the same conditions. Finally, the preparations were mounted in ProLong Gold and incubated for 16 h at room temperature. The analysis of the preparations was done using a BioRad Radiance 2100 laser scanning system on a Zeiss Axiovert 200 microscope. For drug effects on hCLE distribution, HEK293T cells were treated with Actinomycin D at 5 µg/ml in DMEM-10% FBS during 30 min at 37°C. After drug treatment the cells were processed for immunofluorescence as described above.
4
Live Imaging of Ventral Neural Plate
5
Confocal Imaging of Mandible Slices
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Mounted slices were imaged using an upright confocal microscope (Nikon Eclipse FN1 Fixed‐stage with a Radiance 2100 scan head, Zeiss, UK). Dual channel images were collected simultaneously (GFP fluorescence 488 nm excitation and 530/30 emission; and reflection at 488 nm of the cell‐matrix tissue (a polarizer was used to remove mottle)). A simple LUT was used to ensure that there was no saturation of signal at both ends of the grey scale levels (ie no pixels at 0 and 255). The x,y position of the data collection was always made with reference to the agarose bead. Z stack images were collected at 2 µm increments with a Nikon Plan Fluor x10, 0.3 NA lens; importantly, the reflection image was used to ensure the collection regimen was always the same, the two‐surface reflection of the coverslip acted as a reference point which ensured that the data was collected from inside the mandible slice toward the coverslip, this enabled the location of similar depth comparison between DAY 1 and DAY 7. Mandible slices were imaged at both 1 and 7 days after injection with cGFP‐DPPCs (n = 3). Correction of z‐focus distortion was adopted to calculate actual depth of each z‐region, the correction factor in this case was a ratio of the refractive index air:CyGel (i.e., 1.365) 21
6
Umbilical Venous Calcium Dynamics
7
GnRH Neural Projections Analysis
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Fluorescence expression was initially assayed using a Zeiss Axioplan 2 microscope (Carl Zeiss MicroImaging, Inc., Thornwood, NY, USA). For more detailed analysis of fluorescent protein expression and projections, larvae were mounted in 1.0% low-melt agarose with tricaine and imaged with a Zeiss Radiance 2100 laser scanning system together with Laser-Sharp and LSM imaging software (Carl Zeiss MicroImaging, Inc.). Images were processed using Adobe Photoshop CS2. The extensive GnRH neural projections were, at times, obscured by skin or eye autofluorescence when viewed as confocal projections. Therefore, for Figures 5 and 6 , we used ImageJ (http://rsb.info.nih.gov/ij ; developed by Wayne Rasband, NIH) to manually edit each z-slice and remove autofluorescence and background fluorescence, taking care always to spare nearby axons. Movies were made with Fluorender software (http://www.sci.utah.edu/software/46-documentation/137-fluorender.html ). Reconstructed three-dimensional images were projected to a standardized view for figure presentation. Projection lengths were measured using ImageJ on each 100 μm section and were added together for each individual pituitary.
8
Visualizing MCC and PSC Differentiation
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MCC differentiation was assessed in embryos using confocal microscopy to visualize cell boundaries marked with mRFP, Hyls1-GFP, or Chibby-GFP to label basal bodies and staining with a mouse antibody directed against acetylated tubulin (1:1000; Sigma) followed by a cy5 secondary to label cilia. Embryos were processed by a short 10-min fix in 3.7% formaldehyde and 0.25% glutaraldehyde in PBT (PBS with 0.1% TritonX-100) followed by antibody staining. PSC differentiation was analyzed in embryos injected with mGFP RNA followed by staining with a mouse monoclonal anti-AE1 (1:250; Iowa Developmental Studies Hybridoma Bank [IDSHB] clone IVF-12) and rabbit anti-Atp6V1B1/2 (1:100; Santa Cruz Biotechnology) as described previously (Quigley et al. 2011 (link)). Embryos were mounted in PVA with DABCO and imaged on a Bio-Rad Radiance 2100 or Zeiss LSM710 confocal microscope.
Data for basal body counting typically were collected from two to three randomly chosen fields from five to 10 embryos per sample. Cell type number, basal body number, and cell size were quantified using ImageJ software. Statistical significance was performed in all experiments using two-tailed t-tests.
Data for basal body counting typically were collected from two to three randomly chosen fields from five to 10 embryos per sample. Cell type number, basal body number, and cell size were quantified using ImageJ software. Statistical significance was performed in all experiments using two-tailed t-tests.
9
Immunofluorescence Staining of Cultured Cells
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Cells grown on coverslips were fixed using 4% PFA for 20 minutes at room temperature (RT). Cells were permeabilized and blocked using a buffer containing 5% Horse/Donkey/Goat serum, 3% BSA and 0.3% TritonX-100 for 30 minutes at RT. Primary antibodies diluted in antibody diluent (1% Horse/Donkey/Goat serum + 3% BSA + 0.1% TritonX-100) were incubated overnight at 4°C. Secondary antibodies were incubated for 1h at RT. Cells were washed three times with PBS + 0.1%Tween-20. Hoechst (4 μg/mL) was used to visualize nuclei. Coverslips were mounted on slides using Prolong Gold mounting medium (Life Technologies). Images were acquired using confocal microscope Radiance 2100 (Zeiss, Germany) equipped with an upright microscope (Eclipse E800; Nikon, Japan). The Supplementary Table 5 brings a detailed description of the antibodies used in this study.
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