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Imaging of viroids in nuclei from tomato leaf tissue by in situ hybridization and confocal laser scanning microscopy technology (CAT#: STEM-MIT-0140-LJX)

Introduction

The intracellular localization of viroids has been investigated by viroid-specific in situ hybridization and analysis by digital microscopy of the distribution of the fluorescent hybridization signals. Isolated nuclei from green leaf tissue of tomato plants infected with potato spindle tuber viroid (PSTVd) were bound to microscope slides, fixed with formaldehyde and hybridized with biotinylated transcripts of cloned PSTVd cDNA. The bound probe was detected with lissamine--rhodamine conjugated streptavidin. Nucleoli were identified by immunofluorescence using the monoclonal antibody Bv96 and a secondary FITC-conjugated antibody. The distribution of the fluorescence hybridization signals was studied with a confocal laser scanning microscope (CLSM).




Principle

Laser scanning confocal microscope is a high-tech microscope. It is based on fluorescence microscope imaging and equipped with a laser scanning device, which uses ultraviolet or visible light to excite the fluorescence probe, thereby obtaining fluorescence images of the internal microstructure of cells or tissues.
The laser beam is used as the light source in the laser scanning confocal microscope. The laser beam passes through the illuminating pinhole and is reflected to the objective lens through the spectroscope. The laser beam is focused on the sample, and every point on the focal plane of the specimen is scanned. If there is a fluorescent substance that can be excited in the tissue sample, the fluorescence emitted after excitation is directly reversed back to the spectroscope through the original incident light path, and is first focused when passing through the detection pinhole. The focused light is detected and collected by the photomultiplier tube (PMT), and the signal is sent to the computer, and the image is displayed on the computer monitor after processing.

Applications

Imaging and analysis in the fields of morphology, molecular cell biology, neuroscience, pharmacology, genetics

Procedure

1. Sampling
2. Preparation of slices
3. Staining (Select according to the specific experimental situation)
4. Observation

Materials

• Sample Type:
Tomato leaf tissue

Notes

Operate in strict accordance with the operating procedures, and shall not arbitrarily change the operating procedures
In the starting sequence of the switch and in the scanning process, try to do fast and orderly, to protect the laser
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