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A method for preparing carp muscle extracellular connective tissue scanning electron microscope samples

A technology for muscle cells and connective tissue, which is applied in the field of microscopic observation of samples, can solve the problems of incomplete and unclear scanning electron microscope photos, and achieve the effects of shortening drying time, clear structure and uniform penetration

Active Publication Date: 2020-07-14
ZHEJIANG OCEAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The above preparation method is simple, easy to operate, high sperm integrity rate, moderate distribution density, and low technical cost, but this preparation method will dissolve or partially dissolve the connective tissue with the cytoplasm, making the scanning electron microscope photos incomplete or unclear

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  • A method for preparing carp muscle extracellular connective tissue scanning electron microscope samples

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Embodiment 1

[0016] A method for preparing a scanning electron microscope sample of carp muscle extracellular connective tissue, comprising pre-fixation, post-fixation, dehydration, cutting and replacement, freeze-drying, and observation. The specific steps of preparation are:

[0017] 1) Anterior fixation: Cut carp back muscles into 5×5×10 mm pieces avoiding the diaphragm, put them in the prefixative solution for 4 days, replace the fixative solution every 24 hours, avoid the sample, and suck out the prefixation solution with a rubber dropper solution, add 1.8M NaOH solution and shake for 4 days, replace the NaOH solution every 22 hours, avoid the sample, suck out the NaOH solution with a rubber dropper, and then wash it with 0.11M phosphate buffer solution for 10 hours to obtain the pre-fixed sample. The paraformaldehyde concentration in the fixative solution is 2.1%, the glutaraldehyde concentration is 2.3%, the phosphate buffer concentration is 0.11M, and the pH of the pre-fixation solu...

Embodiment 2

[0024] A method for preparing carp muscle extracellular connective tissue scanning electron microscope samples, the specific steps of preparation are:

[0025] 1) Cut the back muscles of carp into pieces avoiding the diaphragm, put them in the pre-fixative solution for 5 days, replace the fixative solution every 23 hours, then suck out the pre-fixative solution, add a solution containing 2.1M NaOH and 0.01mM cyanuric chloride and shake for 3 days , replace the NaOH solution every 23 hours, suck out the NaOH solution, and then wash it with 0.09M phosphate buffer for 14 hours to obtain the pre-fixed sample. The buffer concentration is 0.09M, and the pH of the pre-fixation solution is 7.3. NaOH and cyanuric chloride can cooperate with each other to completely remove proteoglycans and collagen outside the connective tissue cells, and effectively reduce impurities such as extracellular matrix and elastic fibers. The impact on connective tissue, thereby improving the definition of t...

Embodiment 3

[0032] A method for preparing carp muscle extracellular connective tissue scanning electron microscope samples is as follows:

[0033] Cut the back muscles of carp into 5×5×10mm pieces avoiding the diaphragm, put them in the pre-fixation solution for 5 days, and replace the fixation solution every 24 hours. The concentration of paraformaldehyde and glutaraldehyde in the pre-fixation solution are 2% and 2.5% , The concentration of phosphate buffer solution is 0.1M, the pH of the pre-fixative solution is 7.2, then suck out the pre-fixative solution, add 2M NaOH solution, replace the NaOH solution every 24h, shake for 3 days, suck out the NaOH solution, and then wash with 0.1M phosphate buffer solution 12h, the pre-fixed sample was obtained; the pre-fixed sample was fixed in 2% tannic acid solution for 2h, then washed with 0.1M phosphate buffer for 10min, washed repeatedly for 3 times, and then washed with 1% osmium tetroxide Fix for 2 hours, wash with 0.1M phosphate buffer solut...

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Abstract

The invention discloses a method for preparing a scanning electron microscope sample of carp muscle extracellular connective tissue. The method comprises fixing muscle in a pre-fixation solution, sucking out the pre-fixation solution, adding a NaOH solution to the muscle, carrying out oscillation and cleaning, fixing a sample in a tannic acid solution, carrying out cleaning, fixing the sample through osmium tetroxide, carrying out cleaning, washing the sample through a cryoprotectant solution, orderly carrying out dehydration through methanol according to concentration gradient from low to high, putting the dehydrated sample into a methanol solution, carrying out quick-freezing through liquid nitrogen, slicing the sample, carrying out replacement through tertiary butanol, carrying out freeze drying on the sample, and recording the dried sample through photograph. The method can guarantee the complete structure of the sample subjected to cytoplasm removal, does not dissolve or partiallydissolve the connective tissue under action of cytoplasm and keeps the integrity of the connective tissue. Through the method, clear and strong three-dimensional effect images of the connective tissue under scanning electron microscope observation are obtained and the micro-structure characteristics can be well observed.

Description

technical field [0001] The invention relates to the technical field of microscopic observation samples, in particular to a method for preparing carp muscle extracellular connective tissue scanning electron microscope samples. Background technique [0002] Scanning Electron Microscope (SEM) has been widely used in biology, medicine, chemistry, environment, materials, petroleum geology, minerals and many other fields. With the continuous improvement of the resolution of the scanning electron microscope and the gradual improvement of the sample preparation technology, it has played a huge role especially in biological research and has important practical value. In terms of botany, observe leaf epidermis, seeds, pollen, robe sacs and robes, etc., solve morphological anatomy and classification problems, and study the impact of genetic engineering on plants; in plant pathology, observe the morphology, growth status and infecting hosts of pathogenic bacteria The process of observi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N23/2202
CPCG01N23/2202
Inventor 梁佳
Owner ZHEJIANG OCEAN UNIV
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