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Imaging of hippocampal neurons intracellularly labeled with biocytin by laser scanning confocal microscope technology (CAT#: STEM-MIT-0091-LJX)

Introduction

Neurons in slices of rat hippocampus were filled with biocytin and reacted sequentially with avidin-horseradish peroxidase and nickel-intensified diaminobenzidine (DAB/Ni). In all parts of the neuron the DAB/Ni reaction product produced a strong reflection signal in the confocal microscope. The stereo images revealed aspects of three-dimensional hippocampal cell morphology such as the conical shape of the dendritic fields and a characteristic branching pattern of the axon. <br />Labelling neurons intracellularly is an established technique for identifying physiologically-characterized neurons. Recently, confocal microscopy has become a powerful method for examining the three-dimensional morphology of biological specimens.




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:
Hippocampal neurons of the rat

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