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Pretreatment method for integral immunostaining of adult diaphragm

A technology of immunostaining and diaphragm, applied in the preparation of test samples, etc., can solve the problems of limited understanding, difficulty in immunostaining, poor antibody permeability, etc., and achieve the effect of expanding space, fast pretreatment method, and easy removal

Active Publication Date: 2021-03-05
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, knowledge of intact NMJ structural patterns in adulthood remains limited as the diaphragm thickens and is covered by dense connective tissue (Sefton et al., 2018), resulting in poor antibody permeability and less amenable to immunostaining

Method used

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  • Pretreatment method for integral immunostaining of adult diaphragm
  • Pretreatment method for integral immunostaining of adult diaphragm
  • Pretreatment method for integral immunostaining of adult diaphragm

Examples

Experimental program
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Effect test

Embodiment 1

[0034] A device for pre-treatment of whole-mount immunostaining of adult diaphragms, such as figure 1 As shown in A, it includes at least two plates, at least two of which have at least one flat surface, and each plate is provided with at least two through holes.

[0035] In use, place the flat surface of the tablet in contact with the adult diaphragm.

[0036] Generally, a 5 cm x 5 cm plate is applied to the diaphragm of all adult mice. The size of the flat plate can also be adjusted accordingly according to the size of the diaphragm of the animal, and the size of the flat plate is not limited in the present invention.

[0037]In some preferred modes, the flat plate can adopt various shapes, such as rectangle, circle, rhombus, other polygons, etc. No matter what the shape is, it is necessary to ensure that the flat plate covers the diaphragm and the diaphragm cannot be exposed from the flat plate.

[0038] In some preferred manners, the through holes may be screw holes or s...

Embodiment 2

[0044] All mice in the experiment were raised under standard conditions, with 12 hours of light and 12 hours of darkness, with free access to food and water. All animal experimental procedures were approved by the Animal Care and Use Committee of Zhejiang University.

[0045] Obtaining the diaphragm: cut the abdominal skin of the mouse to enter the abdominal cavity, cut the falciform ligament from the diaphragm, separate the liver from the diaphragm, grasp the foot muscle of the diaphragm with tweezers, and gently pull it outward , to separate the diaphragm. The diaphragm can be dissected along the dorsolateral wall with curved dissecting scissors. The dissected diaphragm is cleaned in PBS to remove blood and slightly trimmed by dissecting calf muscle and adipose tissue (Sefton et al., 2018). The diaphragm can also be obtained in other ways in the prior art, and the method of obtaining the diaphragm is not limited in the present invention.

[0046] A pretreatment method for ...

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Abstract

The invention discloses a pretreatment method for integral immunostaining of an adult diaphragm. According to the invention, the space between muscle fibers of adult diaphragm is enlarged by clampingthe diaphragm between two organic glass sheets so as to easily excise connective tissues; and the pressed diaphragm is uniformly expanded, and even if the diaphragm is removed from a device, the diaphragm is still kept flat and expanded, the pressure does not damage the diaphragm, and the thickness of the diaphragm is reduced, so that the whole immunostaining of the adult diaphragm is facilitated,the background color of an immunostaining image is light, and the image is easier to observe.

Description

technical field [0001] The invention specifically relates to a pretreatment method for whole-body immunostaining of an adult diaphragm. Background technique [0002] The neuromuscular junction (NMJ) is a synaptic connection whose three constituent structures are motor neurons, muscle fibers, and terminal Schwann cells (Darabid et al., 2014, Li et al., 2018; Wu et al., 2010 year). Morphological changes in the NMJ are hallmarks of many neuromuscular diseases, such as characteristic amyotrophic lateral sclerosis (ALS), spinal muscular atrophy (SMA), and muscular dystrophy (MD) (Fischer et al.). Pathological features such as axon terminal denervation, swelling, polyinnervation, loss of NMJ complexity, and NMJ disruption are well recognized. These can be easily identified by immunostaining, effectively representing synaptic function. [0003] In a mouse model, some thin, flat muscles such as the diaphragm, transversus abdominis, deltoid sternum, and cranial muscles were staine...

Claims

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Application Information

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IPC IPC(8): G01N1/28G01N1/30
CPCG01N1/28G01N1/30
Inventor 沈承勇杜温佑许文涛张克兢
Owner ZHEJIANG UNIV
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