For microscopy experiments, overnight cultures of C. neoformans strains H99 or H99Nop1-GFP were diluted to 1 × 106 CFU/ml in 3 ml YPD with 2 μg/ml PIK-75 or DMSO (0.02%) and incubated for 24 h at 37°C. Cells were washed three times in sterile PBS, stained with 20 μg/ml CFW in PBS (Fluorescent brightener 28, Sigma, F3543), and incubated for 20 min at RT in the dark. Cells were imaged on a Nikon epifluorescence microscope with a Cool Snap HQ2 camera and Nikon Elements image acquisition and analysis software. Images were processed in Photoshop only to increase ease of viewing. All images were adjusted equally.
Coolsnap hq2
Manufactured by Nikon
Sourced in Japan, United States
The CoolSnap HQ2 is a high-resolution digital camera designed for scientific and industrial applications. It features a 1.4-megapixel CCD sensor and a Peltier cooling system to reduce noise and improve image quality. The camera can capture images with 12-bit depth and supports a variety of data output formats.
Automatically generated - may contain errors
Lab products found in correlation
Metamorph software, by Molecular Devices (4 mentions)
Axiovert 200m, by Zeiss (4 mentions)
Epifluorescence microscope, by Nikon (3 mentions)
Eclipse ti, by Nikon (3 mentions)
Eclipse ti e, by Nikon (3 mentions)
Fluorescent brightener 28, by Merck Group (2 mentions)
Ti microscope, by Nikon (2 mentions)
Softworx suite, by Cytiva (2 mentions)
Em c2 emccd, by Cytiva (2 mentions)
Metamorph 6, by Molecular Devices (2 mentions)
Essentials v 3, by Scientific Volume Imaging (2 mentions)
Eclipse 80i, by Nikon (2 mentions)
Nis elements software, by Nikon (2 mentions)
Lsm 700, by Zeiss (2 mentions)
Plan apo, by Nikon (2 mentions)
Lsm 710, by Zeiss (2 mentions)
Lsm 780, by Zeiss (2 mentions)
Fm4 64, by Thermo Fisher Scientific (1 mentions)
Click it edu assay, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
33 protocols using coolsnap hq2
1
Microscopic Imaging of C. neoformans
2
Fluorescence Microscopy of Bacterial Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Fluorescence microscopy was performed on a Nikon Ti microscope equipped with a plan apo 100×/1.4 NA phase-contrast oil objective and a CoolSnapHQ2 camera. Cells were immobilized using 2% agarose pads containing growth medium. Membranes were stained with FM4-64 (Molecular Probes) at 3 μg/mL. DNA was stained with DAPI at 2 μg/mL. Images were cropped and adjusted using MetaMorph software (Molecular Devices). Final figures were prepared in Adobe Illustrator. Image analysis was performed using MicrobeTracker (Sliusarenko et al. 2011 (link)); details are in the Supplemental Material.
3
Quantifying VSMC Proliferation via EdU Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
We used the Click-iT EdU assay (ThermoFisher) according to the manufacturer’s protocol to identify DNA synthesis in proliferating cells [3 ]. Briefly, VSMCs were seeded on cell culture-treated coverslips at 30% confluence and serum-starved for 18 h. Next, cells were incubated for 24 h in complete growth medium containing EdU reagent (the modified thymidine analog 5-ethynyl-2′-deoxyuridine). Incorporation of EdU was determined by labeling with AlexaFluor azide 647, and total nuclei were labeled with DAPI. Coverslips were mounted and imaged by epifluorescence microscopy (Nikon 80i with CoolSnap HQ2). Four 10× images were obtained per coverslip and the fraction of EdU-positive to total nuclei was determined by using the object count function (Nikon NIS Elements Basic Science). Investigators that captured the images and performed data analysis were blinded to the VSMC genotype.
4
Fluorescence Microscopy of DAPI-Stained Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Fluorescence microscopy was performed on a Nikon Ti microscope equipped with a Plan Apo 100×/1.4 NA phase contrast oil objective and a CoolSnapHQ2 cooled CCD camera. DNA was stained with DAPI (Molecular Probes) at 2 μg/mL. Cells were immobilized using 2% agarose pads containing growth medium. Images were cropped and adjusted using MetaMorph software (Molecular Devices). The final figure preparation was performed in Adobe Illustrator.
5
Immunohistochemical Analysis of Abca4-/- Rdh8-/- Mouse Retina
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Retinal cryosections of Abca4-/- Rdh8-/- mice aged 2 and 18 months were permeabilized with Triton X100 (0.05% in PBS; 5 min at RT) and saturated with NGS (10% in PBS) or BSA (3% in PBS) during 1 h at RT. For cathepsin D immunostaining sections were depigmented in H2O2 (3% in PBS) during 24 h before staining. Primary antibodies against GFAP, RPE65 and cathepsin D were diluted in 2% NGS or 1% BSA and incubated over night at 4°C and followed by Alexa Fluor® 488 - conjugated secondary antibodies during 1 h at RT. In order to quench lipofuscin auto-fluorescence in RPE a final incubation with TrueBlack® was performed. Sections were stained with Hoechst 33342 to label nuclei and representative pictures were taken using a fluorescence microscope (Nikon TiE) equipped with a CoolSNAP HQ2 camera. For each age retinal cryosections from 3 different mice were used.
6
Microscopic Imaging and Analysis of mRNA Distribution
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Epifluorescence micrographs were obtained using an epifluorescence microscope (Nikon TiE) using a Cool Snap HQ2 or QuantEM digital camera. To be able to compare the images shown, fluorescence micrographs from the same wavelengths (Cy3 or Cy5) within an individual experiment were acquired with the same exposure time, and the display scales of the representative images from each condition were equalized. For mRNA distribution analysis, serial Z-sections (0.5-μm steps, between 5 and 7 μm total distances) were acquired, and a maximum projection image was generated using the Nikon Elements software. For quantification of the polarization and distribution of mRNAs, a manual mask was generated using ImageJ, and the dispersion and polarization indices were calculated using the script described (Park et al. 2012 (link)). Cells that contained bright STIC probe aggregates were not imaged for mRNA distribution analysis.
7
Measuring Bacterial Cell Length with Microscopy
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
About 10 μl of cell samples were mounted on a number 1.5, 24 × 50 mm (thickness of 0.16–0.19 mm) cover glass slide (Fisher or VWR). Cells were cushioned with a 3% (w/v) agarose gel pad to restrict the movement of the live cells. Cells were optically imaged using a Nikon Eclipse Ti inverted microscope equipped with crossed polarizers and a Photometrics CoolSNAP HQ2 CCD camera using a Nikon 100X oil objective lens. Phase-contrast images of bacterial cells were recorded with an exposure time of 50 ms using Nikon NIS Elements software. Multiple snapshots were collected for each experiment. All images were analyzed to measure the cell length in Oufti (54 (link)) using one single optimized parameter set and manual verification. Confidence intervals of the fraction of elongated cells for each mutant were computed from 1000 replicates of bootstrap resampling (55 ).
8
Immunofluorescence and Immunoblotting Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were rinsed in PBS, fixed for 10 min in 4% formaldehyde, and permeabilized for 7 min in 0.25% Triton X-100 in PBS. Unless otherwise specified, images were acquired on a Nikon Ti‐E inverted microscope using a CoolSNAP HQ2 camera driven by Nikon Elements software and subsequently deconvolved using the AQI 3D Deconvolution module in Nikon Elements. 2D maximum projections assembled in Elements are shown. Overlays were generated in Photoshop. For immunoblotting, equal numbers of cells were lysed in 2× sample buffer. Proteins were separated by 12% SDS-PAGE, transferred to nitrocellulose, blocked with 5% milk in TBST and probed with primary and secondary antibodies.
9
Quantifying Soluble and Insoluble Aβ42
10
Melanin Production and Laccase Localization in C. neoformans
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!