SMC proliferation was determined with the Click‐iT® EdU Cell Proliferation assay (Invitrogen) according to the manufacturer's protocol. SMCs were serum starved for 18 hours and then seeded at 50% confluence on fibronectin‐coated coverslips in SMC medium supplemented with 5% serum. EdU reagent was added 3 hours after seeding, and the cells were cultured for an additional 24 hours. Samples were gently rinsed with 1% bovine serum albumin in sterile PBS 3 times and fixed with the Click‐iT® fixative solution (15 minutes, room temperature). The samples were gently rinsed twice in 1% bovine serum albumin in sterile PBS, and permeabilized with 1x Click‐iT® sponin‐based permeabilization and wash reagent. EdU‐positive cells were labeled with Cy5 dye by using a click reaction. After excess reagent was rinsed, nuclei were labeled with DAPI. The coverslips were mounted, sealed, and imaged by epifluorescence microscopy on a Nikon 80i microscope equipped with a CoolSnap HQ camera. Four images were captured per coverslip at ×4 magnification. EdU‐positive and DAPI‐positive nuclei were quantified (Nikon NIS Elements software).
Coolsnap hq camera
Manufactured by Nikon
Sourced in United States
The CoolSnap HQ camera is a high-quality, scientific-grade digital camera designed for laboratory and research applications. It features a high-resolution sensor, low noise, and a wide dynamic range, making it suitable for capturing detailed images of a variety of samples and specimens.
Automatically generated - may contain errors
Lab products found in correlation
Nis element, by Nikon (3 mentions)
Lipofectamine rnaimax, by Thermo Fisher Scientific (2 mentions)
Click it edu cell proliferation assay, by Thermo Fisher Scientific (1 mentions)
Nis elements software, by Nikon (1 mentions)
80i microscope, by Nikon (1 mentions)
Rhodamine phalloidin, by Merck Group (1 mentions)
Anti gpsm2, by Merck Group (1 mentions)
E600 microscope, by Nikon (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Eclipse 80i microscope, by Nikon (1 mentions)
Click it edu imaging kit, by Thermo Fisher Scientific (1 mentions)
Nis elements ar 2, by Nikon (1 mentions)
Endomucin, by Santa Cruz Biotechnology (1 mentions)
Phospho histone h3, by Merck Group (1 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
E cadherin, by Cell Signaling Technology (1 mentions)
Vimentin, by Cell Signaling Technology (1 mentions)
Niko digital dx1200me camera, by Universal Imaging (1 mentions)
Lsm880 confocal microscope system, by Zeiss (1 mentions)
8 protocols using coolsnap hq camera
1
Quantifying Smooth Muscle Cell Proliferation
2
Multimodal Adipogenic and Osteogenic Assay
3
Quantifying Phospho-H3 and EdU in FFPE Skin Tissue
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FFPE tissue blocks containing human skin or melanoma tissue were sectioned, deparaffinized, and rehydrated with serial passage through changes in xylene, graded ethanols, and deionized water. All slides were subjected to heat-induced epitope retrieval in target retrieval solution (pH 6.1) (cat S1699; Dako). Slides were then incubated with antiphospho H3 – Alexa488 (#9701; Cell Signaling) for 60 minutes at room temperature in a dark room. EDU staining was done using Click-iT EDU imaging kit following the manufacturer’s instructions (Life Technologies, Cat# C10337). Slides were washed twice in TBS-T, mounted using Vectashield Mounting Medium with DAPI (Vector Laboratories, Cat# H-1500), and analyzed using Leica DMRE Fluorescent microscope and Nikon Coolsnap HQ camera. Phospho H3-positive cells were manually counted from two skin sections. Each section had ~3 mm2 of epidermis surface. Statistical analyses were conducted using a one-way analysis of variance (ANOVA) test with GraphPad Prism software.
4
Immunohistochemical Analysis of Formalin-Fixed Tissues
5
High-resolution Imaging of Murine Skin
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Most samples were analyzed using a Nikon inverted TE2000-E microscope equipped with phase contrast and epifluorescence, a digital CoolSNAP HQ camera, a Prior ProScanII motorized stage, and a Nikon C1 confocal system, using EZC1-3.90 and NIS-Elements -AR acquisition software (Nikon, Melville, NY, USA). Images were acquired with Plan Fluor 4×/0.13, Plan Fluor 10×/0.30, Plan Fluor ELWD 20×/0.45, Plan Apo 40×/1.0 oil, and Plan Apo 100×/1.4 oil objectives, and analyzed with NIS elements (Nikon). Contrast and/or brightness were adjusted for some images to assist in visualization. To image murine skin at high resolution, a Zeiss LSM 880 confocal microscope system with AiryScan detector and a FAST Airyscan module mounted on an AxioObserver (Carl Zeiss, Inc, Peabody, MA, USA)was used. The LSM 880 confocal detection system had 34 spectral-detection channels consisting of a cooled 32 element GaAsP detector array with two flanking photomultiplier tubes (PMTs). Wavelength separation was achieved using high-efficiency grating; maximum spectral resolution was 3 nm over a 286 nm range (410 to 696 nm). Objective lenses: 10×|0.45 NA (air), 20×/0.8 NA DIC, 25×|0.8 multi-immersion DIC, 40×|1.4 NA W DIC, 63×|1.4 NA DIC oil, C-Apochromat 40×/1.2 W/korr FCS, Plan-Apochromat 63×/1.4 NA oil DIC ELYRA. Six single-photon-excitation laser lines were available: 405, 458, 488, 514, 561 and 633 nm.
6
Indirect Immunofluorescence Microscopy Assay
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Cells were cultured, synchronized, and/or infected for indirect immunofluorescence on a μ-Slide VI 0.4 (80606; Ibidi). At various time points, cells were washed twice with cold PBS and fixed with 1% paraformaldehyde (PFA) either for 30 min at room temperature or overnight at 4°C. Immunofluorescence (IF) was assessed as previously described (10 (link)) except to follow manufacturer’s recommendations (AN03 and MV18; Ibidi) for volumes and handling. Images were collected with a Nikon Ti-Eclipse inverted wide-field microscope, taken with a CoolSnap HQ camera, and recorded with Nikon NIS Elements software (v 4.00.03). At least 500 nuclei were counted per condition. Images were processed and background subtracted using NIH FIJI/ImageJ software.
7
Immunofluorescence Imaging of Virus-infected Cells
8
Immunofluorescence of SETD2 in COS7 cells
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COS7 (ATCC, CRL-1651) cells transiently expressing tSETD2-FLAG constructs (Lipofectamine 2000 and OptiMEM) were fixed with 4% formaldehyde in PBS, treated with 50 mM NH4Cl in PBS to quench unreacted formaldehyde and permeabilized with 0.2% Triton X-100 in PBS. Subsequently, cells were blocked in blocking solution (0.2% fish skin gelatin in PBS). Primary antibodies tubulin (DSHB, AB_528499, 1:2000) and FLAG (Abcam, ab205606, 1:2000), and secondary antibodies were applied in blocking solution at room temperature for 1 h each, washing in between with blocking solution. Nuclei were stained with 10.9 μM 4′,6-diamidino-2phenylindole (DAPI) and cover glasses were mounted in ProlongGold (Life Technologies). Cells were incubated 3x for 5 min in blocking solution to remove unbound antibodies. Images were collected on an inverted epifluorescence microscope (Nikon TE2000E) equipped with a 60x, 1.40 numerical aperture oil-immersion objective and a 1.5x tube lens on a Photometrics CoolSnapHQ camera driven by NIS-Elements (Nikon) software.
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