JP3769671B2 - Biological tissue thin section preparation method and adhesive plastic film for thin section support - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to life science studies (eg, histological studies, histochemical studies, enzyme histochemical studies, from biological samples (eg, experimental animal samples, plant samples) embedded (eg, frozen-embedded), A method for preparing a thin section of biological tissue suitable for preparing a thin section (for example, 2 to 10 μm in thickness) that can be used for immunohistochemical research and genetic histochemical research on a slide glass, and adhesiveness for supporting the thin section It relates to plastic films.
[0002]
[Prior art]
Life science research requires thin sections (eg, 2-10 μm thick) of frozen biological samples. However, conventional methods use thin, thin sections (eg, thickened) that can be used for frozen life science studies from large frozen biological specimens (eg, bone-containing specimens, whole body animals, plant samples). 2-10 μm) could not be produced on a slide glass.
[0003]
[Problems to be solved by the invention]
In the conventional method for preparing a thin section of biological tissue, a thin section is easily broken on a slide glass during freezing slicing, and a thin section of a hard tissue or a large biological sample is well maintained with a tissue shape. could not.
[0004]
In order to solve the above-mentioned problems, the present invention can maintain a good tissue form by using an adhesive plastic film for supporting thin slices even at low temperatures (for example, −25 ° C.). The frozen slices are prepared and transferred to a slide glass for use in life science research.
[0005]
[Means for Solving the Problems]
The present invention provides a thin adhesive that adheres to a predetermined surface of a biological sample frozen and embedded in a water-soluble embedding agent even at low temperatures (for example, −25 ° C.) and easily dissolves in an organic solvent. The adhesive plastic film for thin slice support prepared by applying to the plastic film is pasted, and the adhesive plastic film for thin slice support is attached to the sample with the adhesive plastic film for thin slice support with the specified blade. In this state, a thin slice having a predetermined thickness is prepared.
[0006]
Next, the thin section attached to the adhesive plastic film for supporting the thin slice is closely attached to the slide glass, and the slide glass in which the adhesive thin plastic film for supporting the thin section and the thin section is closely attached is immersed in an organic solvent (for example, hexane). The adhesive is dissolved and the plastic film is peeled off from the slide glass to leave only a thin slice on the slide glass.
[0007]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, an embodiment of the present invention will be described with reference to the accompanying drawings.
[0008]
An adhesive plastic film for supporting a thin section provided with a non-adhesive area and an adhesive area, which is used as a support material for a thin section when a frozen sample is sliced, is obtained according to the following first or second embodiment. Make it.
[0009]
-1st Example The preparation procedure of the adhesive plastic film for thin section support in 1st Example is demonstrated with reference to FIGS. 1-5 and FIG. First, as shown in FIG. 1 and FIG. 2 which is a cross-sectional view thereof, on a first plastic film 2 (for example, a polyvinylidene chloride film having a thickness of 10 μm) that is thin, thin, and has low extensibility, at a low temperature ( For example, the adhesive 1 having adhesiveness is applied even at −25 ° C.). After the adhesive 1 is dried, reference numeral 5 in FIG. 1 is set so that the width of the adhesive surface indicated by reference numeral 4 in FIG. 1 is substantially equal to the slicing direction of the frozen sample indicated by reference numeral 15 in FIG. 12A. A second plastic film 3 having a small width indicated by reference numerals 6 and 6 is adhered to the adhesive surface. Next, as shown in FIG. 2, the release paper 7 is brought into close contact with the adhesive surface. As shown in FIG. 12A, the cutting line 8 shown in FIG. 3 is used so that the size of the adhesive surface of the thin section supporting adhesive plastic film is substantially equal to the size of the thin embedded surface of the frozen embedding block 14 during use. Then, the thin film supporting adhesive plastic film for supporting the thin slice shown in FIG. 4 and FIG.
[0010]
-2nd Example The preparation procedure of the adhesive plastic film for thin section support in 2nd Example is demonstrated with reference to FIGS. 6-10 and FIG. First, as shown in FIG. 6, it is adhesive on a first plastic film 2 (for example, polyvinylidene chloride film having a thickness of 10 μm) that is transparent, thin, and has low extensibility, even at a low temperature (for example, −25 ° C.). The pressure-sensitive adhesive 1 is applied in a strip shape with a width substantially equal to the length of the frozen embedding block 14 shown in FIG. After the adhesive 1 is dried, the release paper 7 is brought into close contact with the adhesive surface. As shown in FIG. 12B, the adhesive surface of the thin section-supporting adhesive plastic film is almost equal to the thin cut surface of the frozen embedding block 14 as shown in FIG. 12B. Then, a section-supporting adhesive plastic film shown in FIG. 9 and FIG. 10 which is a cross-sectional view thereof is produced.
[0011]
Next, a biological tissue thin section preparation method for preparing a frozen thin section from a biological sample on a slide glass using the above-described adhesive plastic film for supporting a thin section will be described in order.
i) As shown in FIG. 11A, a biological sample and a water-soluble embedding agent (for example, 5% carboxymethylcellulose gel) are placed in a box-shaped metal container 13 and frozen.
ii) As shown in FIG. 11B, the frozen embedding block 14 is taken out from the metal container 13 and fixed to the frozen slicer.
iii) The thin section supporting adhesive plastic film produced by the above procedure is attached to the sliced surface of the frozen embedding block 14 as shown in FIGS. 12A and 12B.
iv) In this state, the sliced piece is cut with a predetermined blade 17 so that the frozen sliced piece can be picked up by a bear attached to the adhesive plastic film for supporting the sliced piece as shown in FIGS. 13A and 13B.
v) The thin section attached to the adhesive plastic film for supporting the thin section is closely attached to the slide glass 19 that has been surface-treated (for example, silane-coated) as shown in FIG.
vi) As shown in FIG. 15, the slide glass 19 in a state where the thin plastic supporting adhesive plastic film and the thin slice are in close contact with each other is placed in an organic solvent 20 (for example, hexane) placed in a transparent glass container 21. The pressure-sensitive adhesive 1 is dissolved in the upright state, and the plastic film 2 is peeled from the slide glass 19.
vii) As shown in FIG. 16, the slide glass in which only a thin section remains is transferred into 100% ethanol.
viii) The slide glass is then transferred into an aqueous solution.
ix) The thin sections attached to the slide glass are stained according to the research purpose.
[0012]
By the above procedure, the positional relationship of each tissue is maintained from the sample (for example, unfixed non-decalcified hard tissue, brain, whole body of experimental animal, plant) embedded (for example, frozen embedded), and further the tissue A thin section having a thickness of 2 to 10 μm with a good structure can be easily and efficiently produced on a slide glass, and the thin section can be used for life science research.
[0013]
【The invention's effect】
The present invention has the following effects by the methods and materials described above. A biological sample (for example, a sample containing bone) embedded (eg, frozen embedded) by the method described in claim 1 and the thin plastic thin film supporting plastic film described in claim 2. A thin section of 2 to 10 μm in thickness, which has a shape and is not damaged, can be efficiently produced from a laboratory animal's whole body or plant sample), and is attached to the adhesive plastic film for supporting the thin section. In this state, the plastic film is peeled off to leave only a thin slice on the slide glass. Can be efficiently produced on a slide glass.
[0014]
Since the thin section prepared on the slide glass by the method described in claim 1 can be treated with an aqueous solution or an organic solvent (for example, alcohol), many life science studies (for example, Histological research, histochemical research, enzyme histochemical research, immunohistochemical research, gene histochemical research).
[0015]
According to the method described in claim 1, since the thin slice is prepared on a slide glass, it can be stored as a permanent specimen with a conventional mounting medium (for example, Enteran New).
[0016]
The adhesive plastic film for supporting a thin section described in claims 3 and 4 is provided with a region having no adhesive force as a region to be gripped during use, and therefore can be efficiently operated.
[Brief description of the drawings]
FIG. 1 is a plan view of an adhesive-treated plastic film according to an embodiment (first example) of the present invention.
FIG. 2 is a cross-sectional view taken along the line II of FIG.
3 is a plan view showing a cut portion of the adhesive-treated plastic film of FIG. 1. FIG.
4 is a plan view of an adhesive-treated plastic film cut along the cutting line shown in FIG. 3. FIG.
5 is a cross-sectional view taken along line II-II in FIG.
FIG. 6 is a plan view of an adhesive-treated plastic film according to an embodiment (second example) of the present invention.
7 is a cross-sectional view taken along line III-III in FIG.
8 is a plan view showing a cut portion of the adhesive-treated plastic film of FIG. 6. FIG.
9 is a plan view of an adhesive-treated plastic film cut along the cutting line shown in FIG.
10 is a cross-sectional view taken along line IV-IV in FIG.
FIG. 11 is a perspective view showing a box-shaped metal container and a frozen embedding block of a biological sample frozen and embedded in the container.
12 is a perspective view in which the release paper is removed from the thin-section supporting adhesive plastic film of FIGS. 4 and 9, and the thin-section supporting adhesive plastic film is attached to the thin cut surface of the frozen embedding block. FIG.
13 is a cross-sectional view showing a state in which a thin section is being manufactured using the adhesive plastic film for supporting a thin section of FIGS. 4 and 9. FIG.
FIG. 14 is a cross-sectional view showing a state in which a thin section attached to an adhesive plastic film for supporting a thin section is in close contact with a slide glass.
FIG. 15 is a cross-sectional view showing a state in which a slide glass having a thin slice supporting adhesive plastic film and a thin slice in contact with each other is immersed in an organic solvent to dissolve the adhesive.
FIG. 16 is a cross-sectional view showing a state in which the adhesive has dissolved and only a thin section remains on the slide glass.
[Explanation of symbols]
DESCRIPTION OF SYMBOLS 1 ... Adhesive, 2 ... 1st plastic film, 3 ... 2nd plastic film, 4 ... Adhesion area | region, 5 ... Non-adhesion area, 7 ... Non-adhesion area, 7 ... Release paper, 8 ... Cutting line, 9 ... Adhesive area, 10 ... non-adhesive area, 11 ... non-adhesive area, 12 ... cutting line, 13 ... box-type metal container, 14 ... frozen embedding block, 15 ... length of the frozen sample (in the thin slice direction), 16 ... Horizontal length of frozen sample, 17 ... Slicing knife, 18 ... Thin section, 19 ... Slide glass, 20 ... Organic solvent for dissolving adhesive, 21 ... Transparent glass container

Claims (2)

  Adhering an adhesive plastic film for thin section support as a thin section support material to a thin section of a biological sample frozen and embedded with a water-soluble embedding agent, or a biological specimen embedded with paraffin, With the adhesive plastic film for supporting thin slices attached, make a thin slice with a blade, and attach the thin slice attached to the adhesive plastic film for supporting thin slices to a slide glass that has been treated to prevent peeling of the thin slice. Next, immerse the glass slide with the adhesive plastic film for supporting the thin section and the thin section into an organic solvent, dissolve the adhesive, remove the plastic film, and remove the thin section of the biological tissue on the slide glass. A method for preparing a thin section of a biological tissue, characterized in that it is prepared. An adhesive plastic film for supporting a thin section used in the method for preparing a biological tissue thin section according to claim 1,
Its structure is that on one side of a thin and transparent plastic film, an adhesive layer is formed with an adhesive that has strong adhesiveness even at low temperatures and that dissolves easily when immersed in an organic solvent. A thin plastic film is closely attached at a predetermined interval to provide a sticky region and a non-sticky region, and the length of the sticky region sandwiched between the non-sticky regions is the slice of the embedded sample. A pressure-sensitive adhesive sheet is made to be approximately equal to the length in the direction, and a release paper is adhered to the pressure-sensitive adhesive sheet to protect the pressure-sensitive adhesive surface, and the pressure-sensitive adhesive sheet is cut into a thin-cut surface size during use. An adhesive plastic film for supporting thin slices, wherein the release paper is removed from the thin film.

Images