Dyeing method of human craniocerebral specimen grey matter

Abstract

The invention relates to the field of neuroanatomy research, and discloses a human craniocerebral specimen grey matter dyeing method which comprises the following steps: S100, putting a fresh craniocerebral specimen into a neutral formalin solution to fix the specimen; s200, the craniocerebral specimen is soaked in a solution containing 2% of phenol, so that white matter in the craniocerebral specimen generates resistance to dye used subsequently, and a pretreated specimen is obtained; s300, transferring the pretreated specimen into a staining solution containing 0.5% of copper sulfate to perform staining treatment so as to obtain a stained specimen; and S400, washing the stained specimen, and carrying out multi-step dehydration treatment to obtain the stained brain section. According to the method, the grey matter dyeing effect in the brain specimen section is remarkably improved, the contrast of the white matter dyeing effect and the grey matter dyeing effect is strong, observation is easy, and the method is simple, easy to implement and suitable for large-scale neuroanatomy research.

Description

Technical Field

[0001] The invention relates to the field of neuroanatomy research, and in particular to a method for staining gray matter of human cranial brain specimens. Background Art

[0002] In neuroanatomy and pathology research, distinguishing gray matter from white matter is a key step. Traditional staining methods focus on staining white matter fibers, while effective techniques for staining gray matter are relatively scarce, especially when dealing with human cranial specimens, where the staining effect is often limited.

[0003] Anatomical Research, Vol. 23, No. 2, 2001, pp. 185-186, provides a method for preparing and staining brain slice specimens. First, the specimen is placed in 1% ferric chloride for 2 minutes, and the gray matter turns bright gray while the white matter remains unstained. Then, the specimen is placed in 1% potassium ferrocyanide for 3 to 5 minutes, and the gray matter turns blue before being taken out.

[0004] However, the problems with this method are: the coloring reaction is relatively strong and difficult to control. During the staining process, if the time or concentration is not properly controlled, the white matter is easily stained, resulting in unclear boundaries between the two and difficulty in observation; and when coloring, it is easy to color too dark and needs to be placed in warm water for 4 to 6 hours to fade, and the steps are cumbersome.

[0005] Therefore, developing a staining method that can clearly display gray matter structure has important scientific and clinical significance. Summary of the invention

[0006] The purpose of the present invention is to provide a method for staining gray matter of human cranial brain specimens, so as to solve the technical problem in the prior art that the contrast between white matter and gray matter in the specimens is not high after staining.

[0007] In order to solve the above technical problems, the present invention specifically provides the following technical solutions:

[0008] The present invention provides a method for staining gray matter of human cranial brain specimens, comprising the following steps:

[0009] S100, place the fresh brain specimen in a neutral formalin solution for specimen fixation to maintain the integrity of the tissue structure, and remove the excess fixative after fixation;

[0010] S200, soaking the brain specimen in a solution containing 2% phenol, so that the white matter in the brain specimen is resistant to the dye used subsequently, to obtain a pretreated specimen;

[0011] S300, transferring the pretreated specimen to a staining solution containing 0.5% copper sulfate for staining, wherein the gray matter on the pretreated specimen is selectively stained blue, and the white matter remains unstained, thereby obtaining a stained specimen;

[0012] S400, washing the stained specimen and performing a multi-step dehydration process to obtain a stained brain slice.

[0013] As a preferred embodiment of the present invention, in S100, the concentration of the formalin solution is 10%, and the fixation time is 48-72 hours.

[0014] As a preferred embodiment of the present invention, in S200, the immersion time of the brain specimen is 12-24 hours.

[0015] As a preferred embodiment of the present invention, in S300, the dyeing time is 2-4 hours.

[0016] As a preferred embodiment of the present invention, in S400, the multi-step dehydration process comprises the following steps:

[0017] The stained specimens were dehydrated in 70%, 80%, 90% and 100% ethanol solutions in sequence, with each step lasting 5-10 minutes.

[0018] As a preferred embodiment of the present invention, the brain specimen is a human brain specimen.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] The present invention uses 10% neutral formalin solution to fix the cranial brain specimen, maintains the integrity of the tissue structure, and prevents the specimen from being deformed or degraded during subsequent processing;

[0021] The present invention uses a 2% phenol solution to pretreat the cranial brain specimen, so as to make the white matter (myelinated axons) resistant to the dye used subsequently, prevent the dye from penetrating into the white matter, thereby enhancing the staining effect of the gray matter. After staining with 0.5% copper sulfate, the staining effects of the white matter and the gray matter are strongly contrasted, which helps to more clearly observe the gray matter structure and its relationship with the surrounding tissues.

[0022] This method is suitable for neuroanatomy research and clinical pathology analysis, is simple and easy, suitable for large-scale neuroanatomy research, and is also suitable for neuroanatomy staining of other animals, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the implementation methods of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for the implementation methods or the description of the prior art. Obviously, the drawings in the following description are only exemplary, and for ordinary technicians in this field, other implementation drawings can be derived from the provided drawings without creative work.

[0024] Figure 1 A schematic flow chart of a method for staining gray matter of human cranial brain specimens is provided for the present invention;

[0025] Figure 2 The present invention provides a real picture of the slice structure of a human cranial brain specimen when it is not stained;

[0026] Figure 3 The present invention provides a physical picture of the slice structure of the human brain after staining in Example 1. DETAILED DESCRIPTION

[0027] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0028] like Figure 1 The present invention relates to a staining method in the field of neuroanatomy research, and in particular provides a method for staining gray matter of human cranial brain specimens, comprising the following steps:

[0029] 1. Specimen fixation: Place the brain specimen in a 10% neutral formalin solution for fixation for 48-72 hours. The purpose of fixation is to maintain the integrity of the tissue structure and prevent deformation or degradation during subsequent processing.

[0030] 2. Rinse: Rinse the tissue with distilled water or an appropriate solution to remove excess fixative.

[0031] 3. Phenol pretreatment: Soak the specimen in a solution containing 2% phenol for 12-24 hours to obtain a pretreated specimen. The purpose of phenol pretreatment is to make the white matter (myelinated axons) resistant to the dyes used subsequently, prevent the dyes from penetrating into the white matter, and thus enhance the staining effect of the gray matter.

[0032] 4. Staining: The specimen is transferred to a staining solution containing 0.5% copper sulfate for 2-4 hours to obtain a stained specimen. The staining solution can selectively stain the gray matter blue, while the white matter remains unstained.

[0033] 5. Rinse: After staining, rinse the tissue with distilled water or an appropriate solution to remove excess dye.

[0034] 6. Dehydration: Use increasing concentrations of ethanol (such as 70%, 80%, 90%, 100%) to dehydrate the stained specimens, each step for 5-10 minutes.

[0035] The brain specimens were fresh human brain specimens, and this method was also applicable to the neuroanatomical staining of other animals.

[0036] In histological staining, phenol pretreatment distinguishes white matter from gray matter by changing the tissue's affinity for the dye. The action of phenol renders white matter resistant to the blue copper sulfate dye, so that the white matter does not absorb the dye, retaining its original white appearance. In contrast, gray matter is not affected by this resistance to phenol and is able to absorb the blue copper sulfate dye, thus appearing bright blue.

[0037] Furthermore, white matter is mainly composed of myelinated axons, and myelin is an insulating layer composed of lipids and proteins, which has a low affinity for dyes. Phenol treatment may enhance the hydrophobicity of myelin sheaths through cross-linking or other chemical means, further reducing the penetration of dyes and keeping white matter white during the staining process.

[0038] Gray matter contains more neuronal cell bodies, dendrites, and unmyelinated axons. Because gray matter has a lower lipid content, it absorbs dye more easily. In the absence of phenol treatment, gray matter absorbs blue copper sulfate dye and appears bright blue.

[0039] The staining technology of the present invention enhances the contrast between white matter and gray matter so that their boundaries can be clearly seen even under microscope observation, which is helpful for studying brain tissue structure and its pathological changes.

[0040] Through the staining method of the present invention, the white matter (myelinated axons) is made resistant to the dyes used subsequently, preventing the dyes from penetrating into the white matter, enhancing the staining effect of the gray matter, and improving the contrast between the white matter and the gray matter, which helps to more clearly observe the gray matter structure and its relationship with the surrounding tissues.

[0041] This method is suitable for neuroanatomy research and clinical pathology analysis, is simple and easy, suitable for large-scale neuroanatomy research, and is also suitable for neuroanatomy staining of other animals, and has broad application prospects.

[0042] The following provides examples:

[0043] Embodiment 1:

[0044] 1. Specimen fixation: Place fresh human brain specimens in 10% neutral formalin solution for fixation for 50 hours.

[0045] 2. Rinse: Rinse the above specimens with distilled water to remove excess fixative.

[0046] 3. Phenol pretreatment: Soak the specimen in a solution containing 2% phenol for 20 hours.

[0047] 4. Staining: Transfer the specimen to a staining solution containing 0.5% copper sulfate for 3 hours.

[0048] 5. Rinse: After staining, rinse the tissue with distilled water or an appropriate solution to remove excess dye.

[0049] 6. Dehydration: Use 70%, 80%, 90%, and 100% ethanol solutions to clean the stained specimens and dehydrate them for 10 minutes each step.

[0050] 7. Observation: Finally, observe the stained brain specimen slices. The white matter should appear white and the gray matter should appear bright blue, so that the two can be clearly distinguished.

[0051] Observation: Finally, observe the stained brain specimen sections.

[0052] Unstained specimens are shown in Figure 2 , after staining, see Figure 3 (They are not the same slice).

[0053] from Figure 3 As can be seen in the figure, the white matter of the brain specimen after staining in Example 1 should appear white, while the gray matter should appear bright blue, so that the two can be clearly distinguished.

[0054] The human brain specimen gray matter staining method of this embodiment can significantly improve the gray matter staining effect in brain specimen sections. The staining effects of white matter and gray matter have a strong contrast and are easy to observe. The method is simple and easy to implement and is suitable for large-scale neuroanatomy research.

[0055] The above embodiments are only exemplary embodiments of the present application and are not intended to limit the present application. The protection scope of the present application is defined by the claims. Those skilled in the art may make various modifications or equivalent substitutions to the present application within the essence and protection scope of the present application, and such modifications or equivalent substitutions shall also be deemed to fall within the protection scope of the present application.

Claims

1. A method for staining gray matter of human brain specimens, characterized in that: The steps include: S100, place the fresh brain specimen in a neutral formalin solution for specimen fixation to maintain the integrity of the tissue structure, and remove the excess fixative after fixation; S200, soaking the brain specimen in a solution containing 2% phenol, so that the white matter in the brain specimen is resistant to the dye used subsequently, to obtain a pretreated specimen; S300, transferring the pretreated specimen to a staining solution containing 0.5% copper sulfate for staining, wherein the gray matter on the pretreated specimen is selectively stained blue, and the white matter remains unstained, thereby obtaining a stained specimen; S400, washing the stained specimen and performing a multi-step dehydration process to obtain a stained brain slice.

2. A method for staining gray matter of human brain specimens according to claim 1, characterized in that: In the S100, the concentration of the formalin solution is 10%, and the fixation time is 48-72 hours.

3. A method for staining gray matter of human cranial brain specimens according to claim 1, characterized in that: In the S200, the immersion time of the brain specimen is 12-24 hours.

4. The method for staining gray matter of human brain specimens according to claim 1, characterized in that: In the S300, the staining time is 2-4 hours.

5. The method for staining gray matter of human brain specimens according to claim 1, characterized in that: In S400, the multi-step dehydration process includes the following steps: The stained specimens were dehydrated in 70%, 80%, 90% and 100% ethanol solutions in sequence, with each step lasting 5-10 minutes.

6. A method for staining gray matter of human cranial brain specimens according to claim 1, characterized in that: The brain specimen is a human brain specimen.

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