Spermatocyte squashes were obtained as described previously (Page et al. 1998 (link), Viera et al. 2002 (link)). After fixation, squashed spermatocytes were rinsed three times in PBS for 5 min, and incubated with mouse anti-SYCP3 (1:100), ACA/CREST serum (1:30) and guinea pig anti-SKAP (1:100) antibodies diluted in PBS at 4 °C overnight. Nuclei spreads were prepared according to the drying-down technique as described in Peters et al. (1997) (link) and immunostained as described in Grey et al. (2009) (link). For studies in HeLa cells, cells were grown on coverslips and transfected with GFP-SKAP overnight. For immunofluorescence, HeLa cells were washed with PBS, fixed in methanol for 10 min and permeabilized in PBS/0.05% Triton X-100/5% fetal veal serum for 1 h. Cells were incubated with primary antibodies (rabbit anti-GFP, 1:1000 and mouse anti-tubulin clone DM1a, 1:500) in blocking solution at room temperature (RT) for 1 h. After washes in PBS containing 0.1% Tween-20 (PBS-T), cells were incubated with secondary antibodies for 1 h at RT and then stained with DAPI (1:2000). Images were recorded with a Zeiss Axioimager Z1 microscope equipped with a Coolsnap HQ2 camera at 1×1 binning with a 100×, 1.3 NA Olympus U-PlanApo objective. Images were imported into Image J for further processing.
Coolsnap hq2
Manufactured by Olympus
Sourced in Japan, United States
The CoolSNAP HQ2 is a high-quality scientific camera designed for a variety of imaging applications. It features a high-resolution sensor, advanced cooling capabilities, and versatile connectivity options. The camera is capable of capturing detailed images and video, making it a valuable tool for researchers and scientists.
Automatically generated - may contain errors
Lab products found in correlation
Ix71 inverted microscope, by Olympus (3 mentions)
Softworx software, by Cytiva (2 mentions)
Uplfln, by Olympus (2 mentions)
Nanoscanz nz400ce nano positioning piezo z stage, by Prior Scientific (2 mentions)
Ix71 applied precision deltavision restoration microscope, by Cytiva (2 mentions)
Axio imager z1 microscope, by Olympus (1 mentions)
Uplanapo, by Olympus (1 mentions)
Vysis cep 7 d7z1 spectrumorange probe, by Abbott (1 mentions)
Prolong gold, by Thermo Fisher Scientific (1 mentions)
Fura 2 am, by Thermo Fisher Scientific (1 mentions)
Deltavision microscope system, by Cytiva (1 mentions)
Bx61 microscope, by Olympus (1 mentions)
Ix81 fluorescent microscope, by Olympus (1 mentions)
Deltavision algorithms, by Cytiva (1 mentions)
Rhodamine phalloidin, by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (1 mentions)
Softworks, by Cytiva (1 mentions)
Deltavision elite, by Cytiva (1 mentions)
Rc 25f, by Warner Instruments (1 mentions)
Origin software, by OriginLab (1 mentions)
18 protocols using coolsnap hq2
1
Immunodetection of Meiotic Proteins
2
FISH Protocol for Chromosome Analysis
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Cells were grown on a glass coverslip and hypotonically swollen in PBS diluted to 20% with tap water for 5 min. Cells were fixed with methanol–acetic acid (3:1) for 5 min and then dried. The coverslip was hardened for 2 h at 70°C. Fixed cells were denatured with 70% formamide in 2×SSC (300 mM NaCl and 30 mM C6H5Na3O7-2H2O, pH 7.0) for 2 min at 70°C, washed with ethanol, and dried. Denatured cells were incubated with predenatured FISH probes for centromeres of chromosome 7 and 12 (Vysis CEP 7 [D7Z1] SpectrumOrange probe and Vysis CEP 12 [D12Z3] SpectrumGreen probe, respectively; Abbott) in CEP Hybridization Buffer (Abbott) for 12 h at 37°C and washed with 50% formamide in 2×SSC and 1×SSC with DAPI. After final washes, cells were mounted with ProLong Gold (Thermo Fisher Scientific). Z-image stacks were captured in 0.2-µm increments on an Olympus IX-71 inverted microscope controlled by DeltaVision softWoRx using ×20 0.75 NA UPLSAPO objective lens (Olympus) with a CoolSnapHQ2 charge-coupled device camera. Image stacks were projected and saved as TIFF files. Fluorescence foci on the DAPI signal were counted using Speckle Inspector of BioVoxxel ToolBox plug-in (http://www.biovoxxel.de/ ) for Fiji (Schindelin et al., 2012 (link)). All cells analyzed were selected from nonoverlapping fields. Each experiment was successfully repeated at least three times.
3
High-Throughput Fluorescence Imaging Protocols
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Fluorescence images were acquired using a high throughput ImageXpress Ultra confocal microscope with a 40× Plan Fluor objective (MDS Analytical Technologies, CA, USA). Alternatively, fluorescence images were acquired using the Olympus IX-81 high-content screening inverted microscope, attached with a Marzhauser Scan IM (120×100) motorized XY stage controlled by Ludl Mac5000 and a Photometrics CCD camera (CoolSNAP HQ2), with DAPI and EGFP filters and using a 20× Plan Apo objective (Olympus, Japan).
4
Fura-2 Calcium Imaging in A7r5 Cells
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A7r5 cells were cultured on 35 mm poly-d-lysine coated glass coverslips (MatTek corp, MA) and incubated in their respective media at 37°C. When 60–70% confluent, A7r5 cells were loaded with 2 μM Fura-2AM (Invitrogen, NY, USA) for 30 min at 37°C. After incubation, cells were washed with PBS and incubated with Ca2+ containing Ringer buffer for 10 min at room temperature (RT) before analysis. The Ca2+ containing Ringer buffer contained (in mM) 120 NaCl, 5.0 KCl, 1.0 MgCl2, 2.0 CaCl2, 5.5 glucose, and 20 HEPES (pH 7.4). In Ca2+ free Ringer buffer, CaCl2 was replaced with equimolar MgCl2. For experiments that required low Na+, Ringer buffer NaCl was replaced with equimolar amount of N-methyl-D-glucamine (NMDG+). The imaging system included inverted epifluorescence microscope (Olympus IX71, PA) connected to a CoolSNAP HQ2 charged coupled device (CCD) camera. Image acquisition was performed using EasyRatioPro calcium imaging system (PTI, NJ).
Changes in cytosolic Ca2+ level were determined from the ratio of Fura-2AM fluorescence emission intensities following excitation at 340 and 380 nm.
Changes in cytosolic Ca2+ level were determined from the ratio of Fura-2AM fluorescence emission intensities following excitation at 340 and 380 nm.
5
High-Resolution Imaging of Mesophyll Nuclei
6
Progenitor Zone Nuclei Quantification
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Images of dissected gonads fixed in 100% ice-cold methanol and stained with DAPI (VECTASHIELD® Antifade Mounting Medium with DAPI) were taken using an Olympus BX61 microscope with a CoolSnap HQ2 camera. Z-stacks with 1 μm increments were performed in the DAPI channel at a 40x magnification. The nuclei were counted by hand using ImageJ2 v2.9.0/1.53t102 (link). First, the PZ was defined as the region adjacent to the distal tip cell ending at the transition zone boundary where two or more crescent shape nuclei per row of germ cells can be observed38 . The stack of images was cropped at the progenitor zone to the transition zone boundary and nuclei were counted.
7
Fluorescence Imaging of Worms
8
Fluorescence Imaging of Drug Release
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The images were acquired by Photometrics CoolSNAP HQ2 camera mounted on an Olympus iX81 fluorescent microscope. For the red channel a cube comprising a ET560/40x bandpass excitation filter, ET630/75m bandpass emission filter and T585lpxr dichroic filter was used. For the NIR channel a cube comprising a ET740/40x bandpass excitation filter, ET780lp long pass emission filter and T770lpxr dichroic filter was used. To monitor drug release, PANC-1 cell line was grown in six-well culture plates, twice washed with PBS (pH 7.4) and then incubated 10 min with 5-CLB 10 μM solution in PBS containing 2.5% DMSO. After incubation, the samples were twice washed thoroughly with PBS pH 7.4 to remove excess conjugate and the fluorescence changes immediately measured by using fluorescent microscope. All images were taken over time at 37 °C under 6% CO2 atmosphere. Concentration of cells in the imaging experiments was ∼1 × 104 cell per mL. The images were taken in the red and NIR channels. About 10–12 cells were in the entire field of view for each image. The fluorescence intensities of cells were quantified as the integrated density of the full image using ImageJ software.18 The imaging experiments were carried out in triplicate.
9
Fluorescent Imaging of Cytoskeletal Changes
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Cells (2 × 105 cells/well) were seeded in a 6-well plate and incubated overnight. Then cells were treated with the tested compounds for 72 h. After washing with ice-cold PBS, cells were stained with DAPI (Beyotime, United States) and rhodamine-phalloidin (1:200, Sigma, MO, United States) to mark the cytoblast and cytoskeleton, respectively. Pictures were acquired by Photometrics CoolSNAPHQ2 CCD camera on the Olympus IX71-Applied Precision Delta Vision restoration microscope (Applied Precision, United States) and deconvolved using Delta Vision algorithms (Applied Precision, United States).
10
Multicolor Live-Cell Imaging of Yeast
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Freshly growing cells on YES plates were prepared on glass slides with yeast minimum media. Imaging was carried out with a DeltaVision Elite (Applied Precision) comprising an Olympus IX71 inverted fluorescent microscope, and Olympus UPlanSApo 100×, NA1.40, oil immersion objective and a CoolSNAP HQ2 camera. mCherry, YFP and Cerulean signals from cells were captured with 0.2 s (32% filter), 0.4 s (32% filter) and 0.3 s (32% filter) exposures per plane at a 0.3 μm step size over 11 focal planes (z-sections), respectively. These images were deconvolved and analysed using SoftWoRks (Applied Precision). Cell images at the original focal point were also captured using DIC (differential interference contrast) as a reference.
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