A 3D fluorescence in situ hybridization method of poplar root tips based on paraffin sections

Abstract

The invention discloses a poplar root tip 3D fluorescence in-situ hybridization method based on paraffin sections, and belongs to the field of molecular cytogenetics. The poplar root tip 3D fluorescence in-situ hybridization method mainly includes the steps that tender root tips of poplar are obtained and fixed with Carnoy fluid; n-butanol gradient solution dehydrating, dipping wax and embedding are carried out; section slicing, section spreading, section baking and dewaxing are carried out; section preparation adopts pectinase, cellulase, pepsin and RNase for treatment; section preparation, probe denaturation and hybridization overnight are carried out; and hybridization signals are detected after incubation with secondary antibodies is carried out. The poplar root tip 3D fluorescence in-situ hybridization method can obtain clear FISH signals of the poplar root tip paraffin sections, and 3D images captured by a laser scanning confocal microscope can accurately reflect the three-dimensional level spatial distribution of a target sequence in cells. The poplar root tip 3D fluorescence in-situ hybridization method can be used in research fields such as chromosomal space occupation ofpoplar, gene expression and analysis of Hi-C sequencing results, and has broad application prospects.

Description

technical field [0001] The invention belongs to the field of molecular cytogenetics, in particular to a 3D fluorescence in situ hybridization (FISH) method of poplar root tips based on paraffin sections, which can be used for molecular cytogenetics and epigenetics research of poplar. Background technique [0002] Fluorescence in situ hybridization (FISH) is a molecular cytogenetics technique developed in the 1980s. After hybridization based on the principle of complementary base pairing, observe under a fluorescent microscope, and perform qualitative, quantitative or localization analysis on the DNA or RNA in the sample to be tested according to the fluorescent signal. In the past 20 years, plant FISH technology has made great progress, especially in terms of probe types, it has developed from the commonly used rDNA and other repetitive sequence-based probes to the current oligonucleotide (Oligo) sequence probes, FISH technology has been widely used in many plant molecular ...

AI Technical Summary

Problems solved by technology

However, most of the FISH techniques in plants are still at the 2D level, and cannot distinguish the real spatial occupancy of chromosomes in cells.

This is mainly because the production methods of plant FISH are mainly smears, pressed sheets, and drip sheets. Hybrid annealing of sequences, but these production methods change the original spatial position of chromosomes in cells, so they cannot visually display the real spatial occupancy of chromosomes on a three-dimensional level

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