Comet assay of the leaves of tomato plants were performed according to the method described by Sakamoto et al.41 (link). The generated images of ethidium bromide-stained comets were taken by using BX61v microscope (Olympus Co., Tokyo, Japan) equipped with a digital CCD camera (Olympus Co., Tokyo, Japan). The captured comets were examined using CASP software (http://www.casp.of.pl/ ).
Digital ccd camera
Manufactured by Olympus
Sourced in Japan
The Digital CCD camera is a high-performance imaging device designed for laboratory and scientific applications. It features a charge-coupled device (CCD) sensor that captures detailed digital images with excellent resolution and sensitivity. The camera is optimized for a wide range of imaging tasks, providing reliable and accurate data capture for research and analysis purposes.
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9 protocols using digital ccd camera
1
Comet Assay for Tomato Plant DNA Damage
2
Histological Analysis of Articular Cartilage
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The knee samples were fixed in 4% paraformaldehyde at 4 °C for 24 h and then decalcified in 10% EDTA (pH 7.4) for 3 weeks. Subsequently, the joint tissues (the medial femoral condyle) were embedded in paraffin and the 5 μm of the paraffin-embedded tissues were stained using hematoxylin–eosin staining (HE), Masson’s trichrome staining (Masson) and alcian blue staining(Alcian blue). Slides were examined using an optical microscope equipped with a digital CCD camera (Olympus, Japan). The severity of the degradation of the articular cartilage was scored according to the Osteoarthritis Research Society International (OARSI) scoring system as previously described[25 (link)].
3
Simultaneous Monitoring of pH and Currents in HEK-293 Cells
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HEK-293 cells were cultured in 35 mm Petri dishes and co-transfected with cDNA encoding mOTOP1 and the pH-sensitive indicator pHluorin. The cells were lifted and plated on poly-D-lysine-coated coverslips after 24 h transfection. To simultaneously record the response of the pH indicator while measuring currents, once whole-cell recording mode was achieved, the cells were lifted in front of an array of microcapillary tubes (Warner Instruments) and imaging was initiated. Cells were illuminated at 488 nM and emission at 510 nm was detected using a U-MNIBA2 GFP filter cube (Olympus). Images were captured at a frame rate of 1/sec using a Hamamatsu digital CCD camera attached to an Olympus IX71 microscope and analyzed using Simple PCI software. The fluorescence intensity of each cell was measured following subtraction of the background fluorescence.
4
Histological Analysis of Liver Samples
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Liver samples were fixed in 4% paraformaldehyde solution at room temperature for 24 h and stained with hematoxylin and eosin using standard techniques. The samples were embedded in paraffin, and 5-µm thick paraffin sections were cut. The paraffin was removed with xylene, rehydrated through an alcohol gradient, and stained with hematoxylin and eosin and observed using light microscopy at magnification, ×200. Images were captured using light microscope linked to a digital CCD camera (Olympus Corp., Tokyo, Japan).
5
Intracellular pH and Calcium Imaging in Taste Cells
6
Oil Red O Staining of Liver Tissue
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For Oil Red O staining, frozen sections were prepared (6 µm) from liver tissue samples (n=3) and fixed in 50% ethanol. Subsequently, sections were stained with Oil Red O (Beijing Noble Rider Technology Co., Ltd., Beijing, China) for 8 min and differentiated with 50% ethanol, rinsed with tap water, and counterstained with hematoxylin. Following a final rinse in tap water, the sections were mounted with glycerin jelly. The sections were photographed using the light microscope linked to a digital CCD camera (Olympus Corporation).
7
OTOP Channel Activity in HEK-293 Cells
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HEK-293 cells were cultured in 35 mm Petri dishes. OTOP channels and the pH-sensitive indicator pHluorin were co-transfected into the cells. After 24 hr, the cells were lifted and plated on poly-D-lysine-coated coverslips at room temperature. The cells were incubated in standard Tyrodes' solutions before experiments, and the pHluorin fluorescence intensity in response to different solutions was measured. All stimulating solutions were modified from the standard Tyrodes' solution, containing 10 mM of different buffer salt based on the pH (CHES for pH 8.5, MES for pH 6.0, Homo-PIPES for pH 5.0, and Acetic acid for pH 5.0 [HOAC] group). Excitation was 488 nm, and emission was detected at 510 nm using a U-MNIBA2 GFP filter cube (Olympus). Images were acquired on a Hamamatsu digital CCD camera attached to an Olympus IX71 microscope using Simple PCI software. The fluorescence intensity of each cell was normalized to its baseline in Tyrodes' solutions (F0) before the first test stimulus was given to the cells.
8
Simultaneous Monitoring of pH and Currents in HEK-293 Cells
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HEK-293 cells were cultured in 35 mm Petri dishes and co-transfected with cDNA encoding mOTOP1 and the pH-sensitive indicator pHluorin. The cells were lifted and plated on poly-D-lysine-coated coverslips after 24 h transfection. To simultaneously record the response of the pH indicator while measuring currents, once whole-cell recording mode was achieved, the cells were lifted in front of an array of microcapillary tubes (Warner Instruments) and imaging was initiated. Cells were illuminated at 488 nM and emission at 510 nm was detected using a U-MNIBA2 GFP filter cube (Olympus). Images were captured at a frame rate of 1/sec using a Hamamatsu digital CCD camera attached to an Olympus IX71 microscope and analyzed using Simple PCI software. The fluorescence intensity of each cell was measured following subtraction of the background fluorescence.
9
DNA Damage Quantification using Comet Assay
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DNA damage was assayed according to Menke et al. [21 (link)] with minor modification. The experiment was performed in a darkroom with dim red light. About 150 mg leaf samples were sliced in 1 mL PBS buffer (160 mM NaCl, 8 mM Na2HPO4, 4 mM NaH2PO4, pH 7) containing 50 mM EDTA on ice with a fresh razor blade. The 30 μL suspension was then taken on regularly used microscopic slides (pre-coated with 1% of agarose in double distilled H2O and dried over night at room temperature) followed by addition of 30 μL 1% agarose solution (42°C). DNA damage was analysed by the comet assay according to the alkali-alkali (A/A) method as described by Menke et al. [21 (link)]. For comet assay, unwinding was done in high alkali for 5 min, and then electrophoresis for 10 min with 21 V, 300 mA in a chamber cooled on ice, followed by a short neutralisation of 3 min in 100 mM Tris–HCl (pH 7.5). To remove the starch grains, the slides were kept for 10 min in 1% Triton prior to dehydration in 70% (2 min) and 96% (5 min) ethanol and air-drying. The gels were then stained with 15 μL ethidium bromide (5 μg ml-1) and immediately used for evaluation. Images were taken using a fluorescence microscope (BX50WI, Olympus, Japan) equipped with a digital CCD camera (Olympus, Japan).
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