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Toluidine blue rapid staining method of brain tissue frozen section

A frozen section and toluidine blue technology, applied in the field of animal anatomy, can solve the problems of complicated steps, long cycle, excessive background staining, etc., and achieve the effects of reducing wrinkles and breakage, shortening staining time, and increasing continuity

Inactive Publication Date: 2019-08-16
HENAN UNIV OF URBAN CONSTR
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The main disadvantage is that the production process of paraffin sections is long, and it often takes 5-6 days from tissue fixation to section observation, and the production of paraffin sections is complicated. An error in a certain link may cause failure of section production, and paraffin sections are processed with toluidine blue. Long staining time is not easy to control, and it is easy to cause excessive background staining or inconspicuous dendritic and axonal structures
[0004] Therefore, it is necessary to study a rapid toluidine blue staining method for frozen sections of brain tissue, which can avoid the shortcomings of long paraffin section production cycle and cumbersome steps, and can also solve the problem of difficult control of toluidine blue staining time and color separation time. Problem with too dark background

Method used

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  • Toluidine blue rapid staining method of brain tissue frozen section
  • Toluidine blue rapid staining method of brain tissue frozen section
  • Toluidine blue rapid staining method of brain tissue frozen section

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Preparation of frozen sections of the brain:

[0033] (1) The experimental bird was anesthetized by intraperitoneal injection of 3.5% sodium pentobarbital;

[0034] (2) Perfuse room temperature normal saline into the carotid artery of one side of the experimental bird, and output it from the other side of the vein until the outflow is light red or transparent;

[0035] (3) After the perfusion is completed, take out the experimental bird's brain, properly trim the brain as required, and place the brain on the sample holder in the required direction;

[0036] (4) Use tin foil to embed the tissue into a cylinder, drip OTC embedding glue, and put it in the refrigerator at -80℃ for quick freezing for 2 minutes;

[0037] (5) Take out (4) The embedded tissue is sliced ​​in a cryostat with a thickness of 10μm. After the patch is placed, it is immediately placed in AF fixative for 3 minutes.

[0038] The concentration of physiological saline for birds is 0.75%, which is prepared by adding ...

Embodiment 2

[0051] This embodiment is different from embodiment 1 in that the thickness of the slice is further reduced, the concentration of toluidine blue is optimized, and the dyeing time is shortened.

[0052] Preparation of frozen sections of the brain:

[0053] (1) The experimental bird was anesthetized by intraperitoneal injection of 4% sodium pentobarbital;

[0054] (2) Perfuse room temperature normal saline into the carotid artery of one side of the experimental bird, and output it from the other side of the vein until the outflow is light red or transparent;

[0055] (3) After the perfusion is completed, take out the experimental bird's brain, properly trim the brain as required, and place the brain on the sample holder in the required direction;

[0056] (4) Use tin foil to embed the tissue into a cylinder, drip OTC embedding glue, and put it in the refrigerator at -80℃ for quick freezing for 2 minutes;

[0057] (5) Take out (4) The embedded tissue is sliced ​​in a cryostat with a thickne...

Embodiment 3

[0071] The difference between this example and example 1 is that because the brain tissue of some animals is small, it is easy to cause the section to shrink and curl when making thin frozen sections. Therefore, the temperature of toluidine blue staining is optimized in this example. So that thicker frozen sections can also obtain better staining results under the same staining time.

[0072] Preparation of frozen sections of the brain:

[0073] (1) The experimental bird was anesthetized by intraperitoneal injection of 3.5% sodium pentobarbital;

[0074] (2) Perfuse room temperature normal saline into the carotid artery of one side of the experimental bird, and output it from the other side of the vein until the outflow is light red or transparent;

[0075] (3) After the perfusion is completed, take out the experimental bird's brain, properly trim the brain as required, and place the brain on the sample holder in the required direction;

[0076] (4) Use tin foil to embed the tissue int...

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Abstract

The invention relates to a toluidine blue rapid staining method of brain tissue frozen section. The method comprises the following steps: appropriately trimming the treated experimental animal brain tissue, placing on a sample holder in a required direction, embedding the tissue as a cylinder by utilizing tinfoil, dropping in OTC embedding glue, and quickly freezing in a refrigerator at -80 DEG C;taking out the frozen tissue section, and then placing in AF stationary liquid to immobilize immediately; performing washing: placing the immobilized frozen section in distilled water to wash for 5-7s; performing toluidine blue staining: placing the washed frozen section in a toluidine blue solution, which is preheated to 50-60, to stain; performing washing: placing the stained section in the distilled water, washing for 8-14s until the flooding is washed; performing dehydrating: placing the section in 80% of alcohol to dehydrate for 1min; performing color separation: placing the dehydrated section in the 95% of alcohol to perform color separation for 20-30s; performing dehydrating: quickly placing the color-separated section in the anhydrous alcohol to dehydrate for 10-20s; and performing transparency and mounting: placing the section in the xylene-alcohol mixed solution for 3-8s, and then placing in the pure xylene for 5-10s, and then performing the mounting.

Description

Technical field [0001] The invention relates to the field of animal anatomy, in particular to a toluidine blue rapid staining method for frozen sections of brain tissue. Background technique [0002] Toluidine blue is a commonly used synthetic dye. The cations in it have a dyeing effect. The acidic substances of the tissue cells are combined with the cations to be dyed. Toluidine blue staining is a kind of Nissl staining. After staining, the Nissl body is clearly distinguishable, the nucleus and nucleolus are obvious, and axons and dendrites can be distinguished, so it is widely used in the staining of neurons. The toluidine blue staining method needs to be completed by paraffin sections. [0003] The general procedure of toluidine blue staining on paraffin sections requires the tissue to be fixed in advance, and paraffin sections are obtained through dehydration, transparency, wax immersion and other steps, and then deparaffinized before staining can be performed. The main advant...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N1/30G01N1/36
CPCG01N1/30G01N1/36
Inventor 谢朝晖张程王莲哲王艳红陈东莹
Owner HENAN UNIV OF URBAN CONSTR
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