Frozen section method for plant tissues and organs

A plant tissue and frozen section technology, applied in the preparation of test samples, sampling devices, etc., can solve the problems of complex operation process, difficulty in obtaining high-quality slices, and different slice conditions, and achieve simple operation and convenient microscopy Observation and analysis, the effect of low cost

Pending Publication Date: 2017-04-05
SOUTHWEST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, frozen section technology is widely used in the research of animal and human tissues and organs. In plants, because plant cells contain relatively hard cell walls and large vacuoles with high water content, frozen plant tissues and organs are hard and form There are many ice crystals, making it difficult to obtain high-quality slices
In recent years, some research reports have discussed the application and methods of frozen section techniques of different tissues and organs in plants, but most of these methods are to fix plant materials with fixatives such as FAA, paraformaldehyde or ethanol, and a certain concentration Glycerin treatment or liquid nitrogen quick-freezing can get better slicing effect. Not only the slicing conditions are different, but also the operation process is relatively complicated, which is difficult for beginners to master.
In addition, it is also difficult to obtain a complete section for plant tissues and organs with large differences in lignification degree between section tissues, large diameter and large area of ​​spongy cells in the pith (such as the mature main stem of Brassica napus)

Method used

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  • Frozen section method for plant tissues and organs
  • Frozen section method for plant tissues and organs
  • Frozen section method for plant tissues and organs

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Example 1 Preparation, staining and observation of young Brassica napus stem slices

[0021] 1. Preparation of slices

[0022] (1) Sample embedding: Turn on the Leica CM1850 cryostat, lower the temperature of the case to -20°C, and after the temperature stabilizes, use a blade to cut out the young inflorescence stems of Brassica napus at the budding stage (fresh weight water content is 85% Above), use absorbent paper to dry the incision tissue fluid and surface moisture of the sample material, and then apply a layer of frozen embedding solution on the sample holder, put the sample on the sample holder and adjust the sample before the frozen embedding solution solidifies. Angle and orientation, and then use the frozen embedding solution to further embed the sample. When embedding the sample, no air bubbles can form around the sample.

[0023] (2) Slicing: Raise the temperature of the cryostat to -8°C and keep it for more than 30 minutes, then fix the sample holder on th...

Embodiment 2

[0028] Example 2 Preparation, staining and observation of middle and lower stem slices of Brassica napus at the final flowering stage

[0029] 1. Preparation of slices

[0030] (1) Material pretreatment: The middle and lower stem materials of Brassica napus at the final flowering stage (the cross-section of which is as follows figure 2 As shown, it can be seen that the degree of lignification among the section tissues is not uniform and the spongy tissue contained in the pith exceeds one-half of the cross-sectional area of ​​the stalk) soak in an ice-water bath for 2-3 days, and keep ice in the ice-water bath.

[0031] (2) Sample embedding: Turn on the Leica CM1850 cryostat, lower the temperature of the case to -20°C, and after the temperature stabilizes, cut off the pretreated stalk with a blade of 3-8mm, and use absorbent paper to dry the incision tissue fluid of the sample material and the moisture on the surface, and then apply a layer of frozen embedding solution on the...

Embodiment 3

[0037] Example 3 Preparation, staining and observation of anther slices in mid-stage flower buds of Brassica napus

[0038] 1. Preparation of slices

[0039](1) Sample embedding: Turn on the Leica CM1850 cryostat and lower the temperature of the case to -20°C. After the temperature stabilizes, apply a layer of frozen embedding solution on the sample holder, and remove it before the frozen embedding solution solidifies. Calyx Brassica napus mid-term flower bud samples (fresh weight water content above 85%) are placed on the sample holder and the angle and orientation of the sample are adjusted, and then the sample is further embedded with frozen embedding fluid. Bubbles form around the sample.

[0040] (2) Slicing: Raise the temperature of the cryostat to -10°C and keep it for more than 30 minutes, then fix the sample holder on the positionable sample head of the microtome, and slice according to the operation manual of the microtome. In the machine box, use the pre-cooled tw...

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Abstract

The invention discloses a frozen section method for plant tissues and organs. After embedded with an tissue freezing medium at the temperature of minus 20 DEG C to minus 25 DEG C, plant tissues and organs are kept at the temperature of minus 6 DEG C to minus 12 DEG C and then sectioned. By the method, high-quality plant tissue and organ sections with plants' original bioactivity maintained can be obtained rapidly. In addition, the method is easy to operate and low-cost, and is especially applicable to tender plant tissues and organs with fresh weight water-content being 85% and above and plant tissues and organs with nonuniform degree of lignifications in section tissues and with the spongy tissues contained in the medulla being over half of the cross-sectional area of stems. If the prepared section is only for common tissue structure observation, the section can be preserved with ice-water bath for over three days and is convenient for long-time microscopic observation and analysis.

Description

technical field [0001] The invention belongs to the technical field of plant material slicing, and relates to a frozen slicing method suitable for plant tissues and organs. Background technique [0002] In the research of botany and cell biology, slice making, especially obtaining high-quality slices is very important. [0003] Frozen section is an experimental technique in which biological tissue is rapidly frozen at low temperature to a certain hardness and then sectioned. Compared with conventional paraffin sections, frozen sections do not require cumbersome operations such as dehydration, transparency, and wax immersion. Biological tissues will not shrink, and it is easy to maintain the original life form of the cell structure. Low cost and other advantages, so it is often used in histochemistry, immunolocalization and in situ hybridization and other research. [0004] At present, frozen section technology is widely used in the research of animal and human tissues and ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N1/06G01N1/42
CPCG01N1/06G01N1/42
Inventor 尹能文柴友荣李加纳练剑平刘雪李威
Owner SOUTHWEST UNIV
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