JP7360283B2 - A method for comparing and observing the location of a specific protein with the geometric characteristics of the stratum corneum itself and/or the location of a specific tissue within the stratum corneum - Google Patents
A method for comparing and observing the location of a specific protein with the geometric characteristics of the stratum corneum itself and/or the location of a specific tissue within the stratum corneum Download PDFInfo
- Publication number
- JP7360283B2 JP7360283B2 JP2019165711A JP2019165711A JP7360283B2 JP 7360283 B2 JP7360283 B2 JP 7360283B2 JP 2019165711 A JP2019165711 A JP 2019165711A JP 2019165711 A JP2019165711 A JP 2019165711A JP 7360283 B2 JP7360283 B2 JP 7360283B2
- Authority
- JP
- Japan
- Prior art keywords
- stratum corneum
- location
- measurement
- measurement step
- image
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 210000000434 stratum corneum Anatomy 0.000 title claims description 151
- 238000000034 method Methods 0.000 title claims description 76
- 210000001519 tissue Anatomy 0.000 title claims description 37
- 102000004169 proteins and genes Human genes 0.000 title claims description 33
- 108090000623 proteins and genes Proteins 0.000 title claims description 33
- 238000005259 measurement Methods 0.000 claims description 127
- 239000011521 glass Substances 0.000 claims description 26
- 238000010186 staining Methods 0.000 claims description 23
- 238000012744 immunostaining Methods 0.000 claims description 21
- 238000011282 treatment Methods 0.000 claims description 19
- 239000012120 mounting media Substances 0.000 claims description 18
- 239000006059 cover glass Substances 0.000 claims description 16
- 230000000052 comparative effect Effects 0.000 claims description 15
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 15
- 239000000853 adhesive Substances 0.000 claims description 13
- 230000001070 adhesive effect Effects 0.000 claims description 13
- 239000003960 organic solvent Substances 0.000 claims description 11
- 239000007850 fluorescent dye Substances 0.000 claims description 6
- CTQNGGLPUBDAKN-UHFFFAOYSA-N O-Xylene Chemical compound CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 claims description 5
- 239000008096 xylene Substances 0.000 claims description 5
- 102000011799 Desmoglein Human genes 0.000 claims description 4
- 108050002238 Desmoglein Proteins 0.000 claims description 4
- 239000007788 liquid Substances 0.000 claims description 4
- 102000005708 Desmoglein 1 Human genes 0.000 description 12
- 108010045579 Desmoglein 1 Proteins 0.000 description 12
- 238000012360 testing method Methods 0.000 description 11
- XUMBMVFBXHLACL-UHFFFAOYSA-N Melanin Chemical compound O=C1C(=O)C(C2=CNC3=C(C(C(=O)C4=C32)=O)C)=C2C4=CNC2=C1C XUMBMVFBXHLACL-UHFFFAOYSA-N 0.000 description 8
- 210000004027 cell Anatomy 0.000 description 7
- 238000007654 immersion Methods 0.000 description 5
- 210000003491 skin Anatomy 0.000 description 5
- 238000010586 diagram Methods 0.000 description 4
- 238000012545 processing Methods 0.000 description 4
- 239000012192 staining solution Substances 0.000 description 4
- 239000003795 chemical substances by application Substances 0.000 description 3
- 238000012795 verification Methods 0.000 description 3
- 239000000427 antigen Substances 0.000 description 2
- 102000036639 antigens Human genes 0.000 description 2
- 108091007433 antigens Proteins 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 238000012137 double-staining Methods 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 229920005990 polystyrene resin Polymers 0.000 description 2
- 230000035939 shock Effects 0.000 description 2
- 238000002791 soaking Methods 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 241000283707 Capra Species 0.000 description 1
- 229930040373 Paraformaldehyde Natural products 0.000 description 1
- WDVSHHCDHLJJJR-UHFFFAOYSA-N Proflavine Chemical compound C1=CC(N)=CC2=NC3=CC(N)=CC=C3C=C21 WDVSHHCDHLJJJR-UHFFFAOYSA-N 0.000 description 1
- 206010040844 Skin exfoliation Diseases 0.000 description 1
- 206010042496 Sunburn Diseases 0.000 description 1
- 239000008186 active pharmaceutical agent Substances 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- PLKATZNSTYDYJW-UHFFFAOYSA-N azane silver Chemical compound N.[Ag] PLKATZNSTYDYJW-UHFFFAOYSA-N 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 238000004040 coloring Methods 0.000 description 1
- 210000000736 corneocyte Anatomy 0.000 description 1
- ZXJXZNDDNMQXFV-UHFFFAOYSA-M crystal violet Chemical compound [Cl-].C1=CC(N(C)C)=CC=C1[C+](C=1C=CC(=CC=1)N(C)C)C1=CC=C(N(C)C)C=C1 ZXJXZNDDNMQXFV-UHFFFAOYSA-M 0.000 description 1
- 230000035618 desquamation Effects 0.000 description 1
- 230000006866 deterioration Effects 0.000 description 1
- 238000007598 dipping method Methods 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 210000001339 epidermal cell Anatomy 0.000 description 1
- 210000002615 epidermis Anatomy 0.000 description 1
- 229960001235 gentian violet Drugs 0.000 description 1
- 230000004807 localization Effects 0.000 description 1
- 229920002866 paraformaldehyde Polymers 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
- 230000007306 turnover Effects 0.000 description 1
- 238000012800 visualization Methods 0.000 description 1
Landscapes
- Investigating Or Analysing Biological Materials (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Description
本発明は特定のタンパク質の存在位置と角層自体の形状的特徴及び／又は角層内における特定組織の存在位置とを対比観察する方法に関する。 The present invention relates to a method for comparing and observing the location of a specific protein with the geometric characteristics of the stratum corneum itself and/or the location of a specific tissue within the stratum corneum.
肌荒れ、日焼けや乾燥により生じる角層の落屑等の肌状態の悪化に関し、コルネオデスモゾームタンパク質の増加が原因の一つとして知られている（特許文献１）。 An increase in corneodesmosomal protein is known to be one of the causes of deterioration of skin conditions such as rough skin, desquamation of the stratum corneum caused by sunburn and dryness (Patent Document 1).
ここで、コルネオデスモゾームタンパク質のうち、特に、デスモグレインが表皮細胞間及び角質細胞間の接着に関与していることが知られている（特許文献２－４）。 Here, among corneodesmosome proteins, desmoglein is known to be particularly involved in adhesion between epidermal cells and between corneocytes (Patent Documents 2 to 4).
そして、特許文献５には、蛍光抗体法により染色したデスモグレインの存在位置を指標とする表皮ターンオーバーの評価方法及び肌状態評価方法が開示されている。 Patent Document 5 discloses a method for evaluating epidermal turnover and a method for evaluating skin condition using the location of desmoglein stained by fluorescent antibody method as an index.
上記の通り、特定のタンパク質の存在位置と角層自体の形状的特徴及び／又は角層内における特定組織の存在位置には、相関関係があることが知られている。 As mentioned above, it is known that there is a correlation between the location of a specific protein and the geometrical characteristics of the stratum corneum itself and/or the location of a specific tissue within the stratum corneum.
前述の先行技術のあるところ、本発明者らは、簡便に、特定のタンパク質の存在位置と角層自体の形状的特徴及び／又は角層内における特定組織の存在位置とを対比観察する手段を見出し、本発明を完成させた。 While the above-mentioned prior art exists, the present inventors have developed a means for easily comparing and observing the location of a specific protein with the geometrical characteristics of the stratum corneum itself and/or the location of a specific tissue within the stratum corneum. The present invention has been completed.
すなわち、本発明は、特定のタンパク質の存在位置と角層自体の形状的特徴及び／又は角層内における特定組織の存在位置とを対比観察する技術を提供することを課題とする。 That is, an object of the present invention is to provide a technique for comparatively observing the location of a specific protein and the geometrical characteristics of the stratum corneum itself and/or the location of a specific tissue within the stratum corneum.
上記課題を解決する本発明は、角層における特定のタンパク質の存在位置を、免疫染色（Ｉｍｍｕｎｏｓｔａｉｎｉｎｇ）により測定する第一測定工程と、
前記第一測定工程後に、前記第一測定工程で用いた角層と同一の角層に対し、組織染色処理により、角層自体の形状的特徴及び／又は角層内における特定組織の存在位置を測定する第二測定工程と、
前記第一測定工程及び前記第二測定工程の測定結果に基づき、同一の角層における、特定のタンパク質の存在位置と角層自体の形状的特徴及び／又は角層内における特定組織の存在位置と、を対比観察する対比観察工程と、
を有する、方法である。
The present invention to solve the above problems includes a first measurement step of measuring the location of a specific protein in the stratum corneum by immunostaining;
After the first measurement step, the same stratum corneum used in the first measurement step is subjected to tissue staining to determine the geometric characteristics of the stratum corneum itself and/or the location of specific tissues within the stratum corneum. a second measurement step of measuring;
Based on the measurement results of the first measurement step and the second measurement step, the location of a specific protein in the same stratum corneum, the shape characteristics of the stratum corneum itself, and/or the location of a specific tissue within the stratum corneum. a contrast observation step of contrasting observation of ,
It is a method having.
角層は、多層構造を形成している。そして、角層は、熱、薬剤、物理的な衝撃によっても、その構造が崩れにくい。
以上の理由から、本発明においては、対比観察の対象として角層を用いることが好ましい。
The stratum corneum forms a multilayered structure. The structure of the stratum corneum does not easily collapse even when exposed to heat, drugs, or physical shock.
For the above reasons, in the present invention, it is preferable to use the stratum corneum as the object of comparative observation.
また、本発明の好ましい形態では、水溶性封入剤を用い、カバーガラスとスライドガラスとの間に配置された前記角層を封入する処理を前記第一測定工程が含み、
前記第一測定工程と前記第二測定工程との間に、中間処理工程を有し、
前記中間処理工程は、前記第一測定工程後に２５℃以上の温水に前記角層の配置されたスライドガラスを浸し、前記角層を崩さずに前記水溶性封入剤を除くことを含む。
Further, in a preferred embodiment of the present invention, the first measurement step includes a process of encapsulating the stratum corneum disposed between a cover glass and a slide glass using a water-soluble mounting medium,
An intermediate treatment step is provided between the first measurement step and the second measurement step,
The intermediate treatment step includes, after the first measurement step, immersing the slide glass on which the stratum corneum is arranged in hot water of 25° C. or higher to remove the water-soluble mounting medium without destroying the stratum corneum.
また、本発明の好ましい形態では、前記水溶性封入剤がＦｌｕｏｒｏｍｏｕｔ－Ｇである。 In a preferred embodiment of the present invention, the water-soluble mounting medium is Fluoromout-G.
また、本発明の好ましい形態では、前記角層を封入するときに、油溶性接着剤を用い、スライドガラスとカバーガラスとを固定する処理を前記第一測定工程が含み、
前記第一測定工程が、スライドガラスとカバーガラスとを有機溶剤に浸漬させ、前記角層を崩さずに前記油溶性接着剤を除くことを含む。
Further, in a preferred embodiment of the present invention, the first measurement step includes a process of fixing a slide glass and a cover glass using an oil-soluble adhesive when encapsulating the stratum corneum,
The first measurement step includes immersing a slide glass and a cover glass in an organic solvent to remove the oil-soluble adhesive without destroying the stratum corneum.
また、本発明の好ましい形態では、前記有機溶剤がキシレンを含む液剤である。 Further, in a preferred embodiment of the present invention, the organic solvent is a liquid agent containing xylene.
また、本発明の好ましい形態では、前記免疫染色（Ｉｍｍｕｎｏｓｔａｉｎｉｎｇ）が蛍光染色であり、
前記対比観察工程が、
特定のタンパク質の蛍光強度と、
前記角層自体の形状的特徴及び／又は角層内における特定組織の存在位置と、
を対比観察することを含む。
Further, in a preferred embodiment of the present invention, the immunostaining is fluorescent staining,
The comparative observation step
Fluorescence intensity of a specific protein and
the geometrical characteristics of the stratum corneum itself and/or the location of specific tissues within the stratum corneum;
This includes comparing and observing.
また、本発明の好ましい形態では、前記第一測定工程が、角層における、デスモグレインの存在位置を測定する工程である。 In a preferred embodiment of the present invention, the first measuring step is a step of measuring the location of desmoglein in the stratum corneum.
本発明によれば、特定のタンパク質の存在位置と角層自体の形状的特徴及び／又は角層内における特定組織の存在位置とを対比観察する技術を提供することができる。 According to the present invention, it is possible to provide a technique for comparing and observing the location of a specific protein with the geometrical characteristics of the stratum corneum itself and/or the location of a specific tissue within the stratum corneum.
以下、本発明の好ましい実施形態について説明するが、本発明の技術的範囲は以下の実施形態に限定されないことは言うまでもない。 Preferred embodiments of the present invention will be described below, but it goes without saying that the technical scope of the present invention is not limited to the following embodiments.
本発明は、角層における特定のタンパク質の存在位置を、免疫染色（Ｉｍｍｕｎｏｓｔａｉｎｉｎｇ）により測定する第一測定工程と、
第一測定工程後に、第一測定工程で用いた角層と同一の角層に対し、組織染色処理により、角層自体の形状的特徴及び／又は角層内における特定組織の存在位置とを測定する第二測定工程と、
第一測定工程及び第二測定工程の測定結果に基づき、同一の角層における、特定のタンパク質の存在位置と角層自体の形状的特徴及び／又は角層内における特定組織の存在位置と、を対比観察する対比観察工程と、
を有する。
The present invention includes a first measurement step of measuring the location of a specific protein in the stratum corneum by immunostaining;
After the first measurement step, the same stratum corneum used in the first measurement step is subjected to a tissue staining process to measure the shape characteristics of the stratum corneum itself and/or the location of specific tissues within the stratum corneum. a second measurement step of
Based on the measurement results of the first measurement step and the second measurement step, the location of a specific protein in the same stratum corneum, the geometrical characteristics of the stratum corneum itself, and/or the location of a specific tissue within the stratum corneum are determined. A comparative observation step of performing comparative observation;
has.
上記形態とすることにより、特定のタンパク質の存在位置と角層自体の形状的特徴及び／又は角層内における特定組織の存在位置とを対比観察することができる。 By adopting the above-mentioned form, it is possible to compare and observe the location of a specific protein and the geometrical characteristics of the stratum corneum itself and/or the location of a specific tissue within the stratum corneum.
以下、対比観察の対象とする角層に関し、より好ましい形態を説明する。 Hereinafter, a more preferable form of the stratum corneum to be subjected to comparative observation will be described.
対比観察の対象とする角層として、例えば、表皮における角層を挙げることができる。 An example of the stratum corneum targeted for comparative observation is the stratum corneum of the epidermis.
角層は多層構造を形成している。そして、角層は、熱、薬剤、物理的な衝撃によっても、その構造が崩れにくい。
以上の理由から、対比観察の対象として角層を用いることが好ましい。
The stratum corneum forms a multilayered structure. The structure of the stratum corneum does not easily collapse even when exposed to heat, drugs, or physical shock.
For the above reasons, it is preferable to use the stratum corneum as the object of comparative observation.
なお、対比観察の対象とする角層は、被験者の皮膚から取得することで用意することができる。
ここで、被験者の皮膚から角層を取得する方法としては、例えば、テープストリッピング法に依り角層を取得する方法を挙げることができる。
Note that the stratum corneum to be subjected to comparative observation can be prepared by obtaining it from the skin of the subject.
Here, as a method of acquiring the stratum corneum from the skin of the subject, for example, a method of acquiring the stratum corneum by a tape stripping method can be mentioned.
次に、本発明における各工程の詳細を説明する。 Next, details of each step in the present invention will be explained.
＜１＞ 第一測定工程
第一測定工程は、角層における特定のタンパク質の存在位置を、免疫染色（Ｉｍｍｕｎｏｓｔａｉｎｉｎｇ）により測定する工程である。
<1> First measurement step The first measurement step is a step of measuring the location of a specific protein in the stratum corneum by immunostaining.
免疫染色は、蛍光染色、色素染色の何れであっても良い。本発明においては、蛍光染色であることが好ましい。 Immunostaining may be either fluorescent staining or dye staining. In the present invention, fluorescent staining is preferred.
本実施例において、免疫染色は、間接法である。
具体的には、本実施例における免疫染色は、標識していない一次抗体を用いて１度目の抗原抗体反応を行い、一次抗体自体を抗原とする別の標識された抗体（二次抗体）をさらに反応させ２回以上の抗原抗体反応を行う方法である。
In this example, immunostaining is an indirect method.
Specifically, in the immunostaining in this example, a first antigen-antibody reaction was performed using an unlabeled primary antibody, and another labeled antibody (secondary antibody) using the primary antibody itself as an antigen was used. This is a method in which the antigen-antibody reaction is performed two or more times by further reacting.
ただし、本発明における免疫染色は直接法であってもよい。
具体的には、本発明における免疫染色は、抗原に直接反応する一次抗体を標識し、抗原抗体反応を１度しか行わない方法とすることもできる。
However, the immunostaining in the present invention may be a direct method.
Specifically, the immunostaining in the present invention can also be a method in which a primary antibody that directly reacts with an antigen is labeled and the antigen-antibody reaction is performed only once.
測定対象とするタンパク質としては、例えば、コルネオデスモゾームを好ましく挙げることができる。中でも、第一測定工程における測定対象は、デスモグレイン１であることが好ましい。 A preferred example of the protein to be measured is corneodesmosome. Among them, it is preferable that the measurement target in the first measurement step is desmoglein 1.
ただし、本発明において、第一測定工程で測定対象とするタンパク質は、測定においてその局在が把握できるものであれば、その種類に特に制限はない。 However, in the present invention, the type of protein to be measured in the first measurement step is not particularly limited as long as its localization can be ascertained in the measurement.
また、本実施例において、第一測定工程は、免疫染色後の角層の観察にあたり、水溶性封入剤を用い、カバーガラスとスライドガラスとの間に配置された角層を封入する処理を含む。
上記処理を行うことで、より簡便に、特定のタンパク質の存在位置と角層自体の形状的特徴及び／又は角層内における特定組織の存在位置とを対比観察することができる。
In addition, in this example, the first measurement step includes a process of encapsulating the stratum corneum placed between a cover glass and a slide glass using a water-soluble mounting medium when observing the stratum corneum after immunostaining. .
By performing the above processing, it is possible to more easily compare and observe the location of a specific protein with the geometric characteristics of the stratum corneum itself and/or the location of a specific tissue within the stratum corneum.
また、本実施例において、第一測定工程は、角層を封入するときに、油溶性接着剤を用い、スライドガラスとカバーガラスとを固定する処理を含む。
上記処理を行うことで、より簡便に、特定のタンパク質の存在位置と角層自体の形状的特徴及び／又は角層内における特定組織の存在位置とを対比観察することができる。
Furthermore, in this example, the first measurement step includes a process of fixing the slide glass and the cover glass using an oil-soluble adhesive when encapsulating the stratum corneum.
By performing the above processing, it is possible to more easily compare and observe the location of a specific protein with the geometric characteristics of the stratum corneum itself and/or the location of a specific tissue within the stratum corneum.
また、第一測定工程の測定結果は、画像として取得する形態であることが好ましい。 Moreover, it is preferable that the measurement result of the first measurement step is acquired as an image.
また、本発明の好ましい実施の形態では、第一測定工程が、スライドガラスとカバーガラスとを有機溶剤に浸漬させ、角層を崩さずに油溶性接着剤を除くことを含む。 In a preferred embodiment of the present invention, the first measurement step includes immersing the slide glass and cover glass in an organic solvent to remove the oil-soluble adhesive without disturbing the stratum corneum.
特に、油溶性接着剤がマニキュアである場合に、有機溶剤に浸漬させ、角層を崩さずに油溶性接着剤を除くことが好ましい。 In particular, when the oil-soluble adhesive is nail polish, it is preferable to immerse it in an organic solvent to remove the oil-soluble adhesive without disturbing the stratum corneum.
また、有機溶剤としては、キシレンを含む液剤を好ましく挙げることができる。 Further, as the organic solvent, a liquid agent containing xylene can be preferably mentioned.
ここで、角層の配置されたスライドガラスへの有機溶剤の適用時間は、１分以上、より好ましくは２分以上である。
下限以上の時間とすることで、より確実に油溶性接着剤を除くことができる。
Here, the time period for which the organic solvent is applied to the slide glass on which the stratum corneum is arranged is 1 minute or more, more preferably 2 minutes or more.
By setting the time to be longer than the lower limit, the oil-soluble adhesive can be removed more reliably.
また、角層の配置されたスライドガラスへの有機溶剤の適用時間は、３０分以下、より好ましくは２０分以下、さらに好ましくは１０分以下である。
上限以下の時間とすることで、角層を壊さずに、油溶性接着剤を除くことができる。
Further, the time period for applying the organic solvent to the glass slide on which the stratum corneum is arranged is 30 minutes or less, more preferably 20 minutes or less, and still more preferably 10 minutes or less.
By setting the time to less than the upper limit, the oil-soluble adhesive can be removed without damaging the stratum corneum.
ここで、有機溶剤の温度は、好ましくは３０℃以下、より好ましくは２５℃以下である。 Here, the temperature of the organic solvent is preferably 30°C or lower, more preferably 25°C or lower.
有機溶剤に浸漬させ、角層を崩さずに油溶性接着剤を除く処理は、温水に角層の配置されたスライドガラスを浸す前に行うことが好ましい。 The treatment of removing the oil-soluble adhesive without destroying the stratum corneum by immersing it in an organic solvent is preferably carried out before dipping the glass slide on which the stratum corneum is arranged in warm water.
＜２＞中間処理工程
中間処理工程とは、第一測定工程後の角層を第二測定工程に供するための準備工程をいう。
<2> Intermediate treatment step The intermediate treatment step refers to a preparation step for subjecting the stratum corneum after the first measurement step to the second measurement step.
水溶性封入剤を用い、カバーガラスとスライドガラスとの間に配置された角層を封入する処理を第一測定工程が含む場合には、中間処理工程として、第一測定工程後に２５℃以上の温水に角層の配置されたスライドガラスを浸し、角層を崩さずに水溶性封入剤を除くことを含むことが好ましい。 If the first measurement step includes a process of encapsulating the stratum corneum placed between the cover glass and the slide glass using a water-soluble mounting medium, as an intermediate treatment step, the Preferably, the step includes immersing the glass slide on which the stratum corneum is arranged in warm water and removing the water-soluble mounting medium without disturbing the stratum corneum.
特に、水溶性封入剤がＦｌｕｏｒｏｍｏｕｔ－Ｇである場合に、中間処理工程として、第一測定工程後に２５℃以上の温水に角層の配置されたスライドガラスを浸す処理を行うことが好ましい。 In particular, when the water-soluble mounting medium is Fluoromout-G, it is preferable to immerse the glass slide on which the stratum corneum is arranged in hot water of 25° C. or higher as an intermediate treatment step after the first measurement step.
ここで、角層を崩さずに水溶性封入剤を除く処理における、温水の温度は、好ましくは２５℃以上、より好ましくは３０℃以上、より好ましくは３５℃以上である。
下限以上の温度とすることで、水溶性封入剤を除くことができる。
Here, the temperature of the hot water in the process of removing the water-soluble mounting agent without disrupting the stratum corneum is preferably 25°C or higher, more preferably 30°C or higher, and even more preferably 35°C or higher.
By setting the temperature above the lower limit, the water-soluble mounting medium can be removed.
また角層を崩さずに水溶性封入剤を除く処理における、温水の温度は、好ましくは４５℃以下、より好ましくは４０℃以下である。
上限以下の温度とすることで、角層を壊さずに、水溶性封入剤を除くことができる。
Further, the temperature of the hot water in the treatment for removing the water-soluble mounting medium without disrupting the stratum corneum is preferably 45°C or lower, more preferably 40°C or lower.
By keeping the temperature below the upper limit, the water-soluble mounting medium can be removed without damaging the stratum corneum.
ここで、温水の温度が２５℃～３０℃の場合の浸漬時間は、好ましくは３日以上、より好ましくは４日以上である。 Here, the immersion time when the temperature of the hot water is 25° C. to 30° C. is preferably 3 days or more, more preferably 4 days or more.
また、温水の温度が３０℃～３５℃の場合の浸漬時間は、好ましくは１日以上、より好ましくは２日以上である。 Further, when the temperature of the hot water is 30° C. to 35° C., the immersion time is preferably one day or more, more preferably two days or more.
また、温水の温度が３５℃～４０℃の場合の浸漬時間は、好ましくは１２時間以上、より好ましくは１６時間以上である。 Further, when the temperature of the hot water is 35° C. to 40° C., the immersion time is preferably 12 hours or more, more preferably 16 hours or more.
また、温水の温度が４０℃以上の場合の浸漬時間は、好ましくは１２時間未満である。 Further, when the temperature of the hot water is 40° C. or higher, the immersion time is preferably less than 12 hours.
上記の条件とすることで、より確実に、角層を崩さずに水溶性封入剤を除くことができる。 By meeting the above conditions, the water-soluble mounting medium can be removed more reliably without disrupting the stratum corneum.
＜３＞ 第二測定工程
第二測定工程は、第一測定工程後に、第一測定工程で用いた角層と同一の角層に対し、組織染色処理により、角層自体の形状的特徴及び／又は角層内における特定組織の存在位置を測定する工程である。
<3> Second measurement process In the second measurement process, after the first measurement process, the same stratum corneum used in the first measurement process is subjected to a tissue staining process to determine the shape characteristics and/or characteristics of the stratum corneum itself. Alternatively, it is a step of measuring the location of a specific tissue within the stratum corneum.
本明細書において、組織染色処理とは、所望の形状的特徴の存在位置を組織学的に染色する処理をいう。 As used herein, tissue staining refers to a process of histologically staining the location of a desired geometric feature.
本明細書において、角層自体の形状的特徴とは、角層そのもの形状、形状的特徴をいう。 In this specification, the shape characteristics of the stratum corneum itself refer to the shape and shape characteristics of the stratum corneum itself.
角層自体の染色処理は、角層の形状的特徴が観察できるものであれば、その手法に特に制限はない。
ここで、角層染色剤としては、例えば本出願人による特開２００６－０５３１１７号公報などに記載された、ゲンチアナバイオレットといった角層染色剤を挙げることができる。
There are no particular limitations on the method of dyeing the stratum corneum itself, as long as the geometrical characteristics of the stratum corneum can be observed.
Here, examples of the stratum corneum stain include a stratum corneum stain such as gentian violet, which is described in JP-A No. 2006-053117 by the present applicant.
また、本明細書において、角層内における特定組織の存在位置とは、生体組織、細胞の構造、機能特徴の存在する位置をいう。 Furthermore, in this specification, the position of a specific tissue within the stratum corneum refers to the position where a biological tissue, cell structure, or functional characteristic exists.
特定組織の染色処理としては、たとえば、フォンタナマッソン染色を挙げることができる。 Examples of staining treatments for specific tissues include Fontana Masson staining.
特定組織としては、染色により観察可能な組織であれば特に制限はなく、例えば、メラニンの局在を挙げることができる。 The specific tissue is not particularly limited as long as it can be observed by staining, and includes, for example, localized melanin.
また、第二測定工程の測定結果は、第一測定工程で取得した画像と同一の部分を画像として取得する形態であることが好ましい。 Moreover, it is preferable that the measurement result of the second measurement step is obtained as an image of the same part as the image obtained in the first measurement step.
＜４＞対比観察工程
対比観察工程は、第一測定工程及び第二測定工程の測定結果に基づき、同一の角層における特定のタンパク質の存在位置と角層自体の形状的特徴及び／又は角層内における特定組織の存在位置を対比観察する工程である。
<4> Comparative observation step The comparative observation step is based on the measurement results of the first measurement step and the second measurement step, and the location of a specific protein in the same stratum corneum, the shape characteristics of the stratum corneum itself, and/or the stratum corneum itself. This is a process of comparing and observing the location of specific tissues within the tissue.
ここで、第一測定工程での免疫染色（Ｉｍｍｕｎｏｓｔａｉｎｉｎｇ）が蛍光染色である場合には、
対比観察工程は、特定のタンパク質の蛍光強度と、角層自体の形状的特徴及び／又は角層内における特定組織の存在位置と、を対比観察することを含む形態であることが好ましい。
Here, if the immunostaining in the first measurement step is fluorescent staining,
Preferably, the comparative observation step includes comparative observation of the fluorescence intensity of a specific protein and the geometrical characteristics of the stratum corneum itself and/or the location of a specific tissue within the stratum corneum.
また、第一測定工程及び第二測定工程の測定結果を対比する方法としては、第一測定工程の測定結果の画像と、第二測定工程の測定結果の画像とを並列に配置し、本分野を専門とする評価者により、特定のタンパク質の存在位置と角層自体の形状的特徴及び／又は角層内における特定組織の存在位置とを対比観察する方法を挙げることができる。 In addition, as a method for comparing the measurement results of the first measurement process and the second measurement process, an image of the measurement results of the first measurement process and an image of the measurement results of the second measurement process are placed in parallel, and One example is a method in which an evaluator who specializes in this compares and observes the location of a specific protein with the geometrical characteristics of the stratum corneum itself and/or the location of a specific tissue within the stratum corneum.
また、第一測定工程及び第二測定工程の測定結果を対比する他の方法として、第一測定工程の測定結果の画像と、第二測定工程の測定結果の画像とを重ね合わせた照合後画像を生成することを挙げることができる。
具体的には、照合後画像生成手段により、第一測定工程の測定結果の画像と、第二測定工程の測定結果の画像とを重ね合わせた照合後画像を生成し、端末に表示可能とすることもできる。
In addition, as another method of comparing the measurement results of the first measurement process and the second measurement process, an image after comparison is obtained by superimposing an image of the measurement results of the first measurement process and an image of the measurement results of the second measurement process. One example is the generation of .
Specifically, the post-matching image generation means generates a post-matching image in which the image of the measurement results of the first measurement process and the image of the measurement results of the second measurement process are superimposed, and the image can be displayed on the terminal. You can also do that.
ここで、照合後画像を生成する方法に特に制限はなく、例えば、第一測定工程の測定結果の画像と、第二測定工程の測定結果の画像の一致率が高くなるよう、画像の向き変更処理、画像の拡大縮小処理をおこなうことで、第一測定工程の測定結果の画像と、第二測定工程の測定結果の画像とを重ね合わせた照合後画像を生成する方法をとることができる。 Here, there is no particular restriction on the method of generating the image after matching. For example, the orientation of the image may be changed to increase the matching rate between the image of the measurement results of the first measurement process and the image of the measurement results of the second measurement process. By performing processing and image scaling processing, it is possible to generate a collated image in which the image of the measurement result of the first measurement step and the image of the measurement result of the second measurement step are superimposed.
中でも、照合後映像を生成する方法は、各染色前の細胞の配置の一致率が高くなるよう、画像の拡大縮小処理をおこなうことで、第一測定工程の測定結果の画像と、第二測定工程の測定結果の画像とを重ね合わせた照合後映像を生成する方法とすることが好ましい。 Among these methods, the method of generating a post-verification image involves scaling the image to increase the matching rate of cell placement before each staining, and then comparing the image of the measurement results from the first measurement process with the image from the second measurement. It is preferable to use a method of generating a post-verification image by superimposing an image of the measurement result of the process.
また、照合後映像における、第一測定工程及び第二測定工程の測定結果の差分を可視化することが好ましい。
具体的には、差分可視化手段により、差分の色付け、差分の透かし、差分の切り出し、差分の印付け、から選ばれる１又は２以上の方法により、照合後映像における、第一測定工程及び第二測定工程の測定結果の差分を可視化し、端末に表示可能とすることもできる。
Moreover, it is preferable to visualize the difference between the measurement results of the first measurement process and the second measurement process in the post-verification video.
Specifically, the difference visualization means performs the first measurement process and the second measurement process in the image after matching using one or more methods selected from coloring the difference, watermarking the difference, cutting out the difference, and marking the difference. It is also possible to visualize the difference between the measurement results of the measurement process and display it on a terminal.
以下、本発明の基礎となる知見を裏付ける各種試験結果を示す。 Below, various test results supporting the findings that form the basis of the present invention will be shown.
＜試験１＞ 角層の同一部位における、デスモグレイン１の免疫染色と、細胞形態染色（角層染色）による二重染色を試みた。 <Test 1> Double staining of desmoglein 1 immunostaining and cell morphology staining (horny layer staining) at the same site of the stratum corneum was attempted.
（１）被検試料の採取
テープストリッピング法により、角層を５１歳男性の腕から採取した。
採取した角層をスライドガラスに貼付した。
(1) Collection of test sample The stratum corneum was collected from the arm of a 51-year-old man by the tape stripping method.
The collected stratum corneum was attached to a glass slide.
（２）第一測定工程：デスモグレイン１の免疫染色
角層を添付したスライドガラスを、キシレン（Ｗａｋｏ 製）に一晩浸漬した。
浸漬後の角層を洗浄、風乾し、４％パラホルムアルデヒド・リン酸緩衝液（Ｗａｋｏ 製）中、室温条件下で１５分間静置した。
(2) First measurement step: Desmoglein 1 immunostaining The glass slide with the stratum corneum attached was immersed in xylene (manufactured by Wako) overnight.
After immersion, the stratum corneum was washed, air-dried, and left standing in a 4% paraformaldehyde phosphate buffer (manufactured by Wako) at room temperature for 15 minutes.
角層をＰＢＳ（Ｗａｋｏ 製）で洗浄した。洗浄後の角層を、２０％ブロックエース水溶液（ＤＳ バイオファーマメディカル株式会社 製）中、室温条件下、１時間静置した。 The stratum corneum was washed with PBS (manufactured by Wako). The washed stratum corneum was left standing in a 20% Block Ace aqueous solution (DS BioPharma Medical Co., Ltd.) at room temperature for 1 hour.
角層を添付したスライドガラス（ＭＡＳ コート ＭＡＴＳＵＮＡＭＩ 製）をスーパーパップペンリキッドブロッカー（大道産業株式会社 製）で枠付けし、角層に一次抗体（Ａｎｔｉ－Ｄｅｓｍｏｇｌｅｉｎ１，Ｍｏｕｓｅ－Ｍｏｎｏ（Ｄｓｇ１－Ｐ２３）（Ｐｒｏｇｅｎ，６５１１１０）原液：ＩｇＧ 濃度：１０－１５μｇ／ｍＬ）を加え、湿潤箱中、室温条件下で２時間静置した。
静置後、ＰＢＳを用い、角層を洗浄した。
A slide glass with the stratum corneum attached (MAS coat, manufactured by MATSUNAMI) was framed with Super Pup Pen Liquid Blocker (manufactured by Daido Sangyo Co., Ltd.), and primary antibodies (Anti-Desmoglein1, Mouse-Mono (Dsg1-P23)) were applied to the stratum corneum. Progen, 651110) stock solution: IgG concentration: 10-15 μg/mL) was added, and the mixture was allowed to stand for 2 hours at room temperature in a humid chamber.
After standing still, the stratum corneum was washed using PBS.
洗浄後、二次抗体（Ａｌｌｅｘａ Ｆｌｕｏｒ＠４８８ Ｇｏａｔ Ａｎｔｉ－ｍｏｕｓｅ ＩｇＧ（Ｉｎｖｉｔｒｏｇｅｎ，Ａ－１１００１ ＰＢＳ２００倍希釈液）を加え、湿潤箱中で室温、遮光条件下で、１時間静置した。
静置後、ＰＢＳ、蒸留水を用い、角層を洗浄した。
After washing, a secondary antibody (Allexa Fluor@488 Goat Anti-mouse IgG (Invitrogen, A-11001 PBS 200-fold diluted solution) was added, and the mixture was allowed to stand for 1 hour at room temperature under light-shielded conditions in a humid box.
After standing still, the stratum corneum was washed using PBS and distilled water.
洗浄後、Ｆｌｕｏｒｏｍｏｕｔ－Ｇ（水溶性封入剤）を用い、角層をスライドガラスとカバーガラスとの間に封入した。封入後、マニキュア（有機接着剤）を用い、角層をスライドガラスとカバーガラスとを固定した。
固定後、乾燥させ、デスモグレイン１の蛍光を観察した。
ここで、観察終了まで、作業時以外は、遮光条件を維持していた。
観察後、キシレンに３分間浸漬させることにより、マニキュア（有機接着剤）を除去した。
After washing, the stratum corneum was mounted between a slide glass and a cover glass using Fluoromout-G (a water-soluble mounting medium). After enclosing, the stratum corneum was fixed to a slide glass and a cover glass using nail polish (organic adhesive).
After fixation, it was dried and the fluorescence of desmoglein 1 was observed.
Here, until the end of the observation, light shielding conditions were maintained except during work.
After observation, the nail polish (organic adhesive) was removed by immersing it in xylene for 3 minutes.
（３）中間処理工程（第一測定工程後、第二測定工程前の処理）
角層を、３７℃蒸留水に一晩浸漬させることにより、Ｆｌｕｏｒｏｍｏｕｔ－Ｇ（水溶性封入剤）を除去し、カバーガラスを外した。
ここで、上記中間処理を経ずにカバーガラスを外した角層は、後の第二測定工程において、角層細胞の形状が崩れてしまうものが散見された。
(3) Intermediate treatment process (treatment after the first measurement process and before the second measurement process)
Fluoromout-G (water-soluble mounting medium) was removed by immersing the stratum corneum in distilled water at 37° C. overnight, and the cover glass was removed.
Here, in the stratum corneum from which the cover glass was removed without undergoing the above-mentioned intermediate treatment, the shape of the stratum corneum cells sometimes collapsed in the subsequent second measurement step.
（４）第二測定工程：細胞形態染色（角層染色） (4) Second measurement step: Cell morphology staining (horny layer staining)
中間処理工程後の角層を、細胞形態染色液（角層染色液 下記表１）に、室温条件下、１０分間浸漬させた。浸漬後、角層を水洗、風乾した。
風乾後の角層をＮｅｗ Ｍ・Ｘ（ポリスチレン樹脂封入剤）でスライドガラスとカバーガラスとの間に封入し、角層の観察を行った。
The stratum corneum after the intermediate treatment step was immersed in a cell morphology staining solution (horny layer staining solution, Table 1 below) for 10 minutes at room temperature. After soaking, the stratum corneum was washed with water and air-dried.
The air-dried stratum corneum was encapsulated between a slide glass and a cover glass using New M.X (polystyrene resin mounting medium), and the stratum corneum was observed.
（５）結果、考察
測定結果を図１に示す。
ここで、第一測定工程の測定結果の画像と、第二測定工程の測定結果の画像とを並列に配置し、本分野を専門とする評価者により、デスモグレイン１の存在位置と、角層の重なりの多い位置とを対比観察した。
(5) Results and discussion The measurement results are shown in Figure 1.
Here, the image of the measurement results of the first measurement process and the image of the measurement results of the second measurement process are arranged in parallel, and an evaluator who specializes in this field determines the location of desmograin 1 and the stratum corneum layer. We compared and observed the positions where there was a lot of overlap.
図１に示す通り、蛍光免疫染色によりデスモグレイン１の存在位置を測定する第一測定工程を行った後、角層の重なりの多い位置を細胞形態染色液（角層染色液）を用い測定する第二測定工程を行うことで、同一の角層におけるデスモグレイン１の存在位置と角層における角層の重なりの多い位置を、効率よく対比観察できることがわかった。 As shown in Figure 1, after performing the first measurement step of measuring the location of desmoglein 1 using fluorescent immunostaining, the location where the stratum corneum overlaps a lot is measured using a cell morphology staining solution (horny layer staining solution). It has been found that by performing the second measurement step, it is possible to efficiently compare and observe the location of desmoglein 1 in the same stratum corneum and the location in the stratum corneum where the stratum corneum overlaps a lot.
ここで、デスモグレイン１は、染色により、細胞膜の輪郭を示すことのできるタンパク質である。すなわち、デスモグレイン１は、第一測定工程によりそのタンパク質の存在位置が把握できるものである。 Here, desmoglein 1 is a protein that can show the outline of a cell membrane by staining. That is, desmoglein 1 allows the location of the protein to be determined by the first measurement step.
以上の通り、免疫染色（Ｉｍｍｕｎｏｓｔａｉｎｉｎｇ）により特定のタンパク質の存在位置を測定する第一測定工程を行った後、角層自体の形状的特徴を染色処理により測定する第二測定工程を行うことで、同一の角層における特定のタンパク質の存在位置と角層自体の形状的特徴を測定することができることがわかった。 As mentioned above, after performing the first measurement step of measuring the location of a specific protein by immunostaining, by performing the second measurement step of measuring the shape characteristics of the stratum corneum itself by staining, We found that it is possible to measure the location of specific proteins in the same stratum corneum and the geometrical characteristics of the stratum corneum itself.
＜試験２＞ 角層の同一部位における、デスモグレイン１の免疫染色と、フォンタナマッソン染色の二重染色を試みた。 <Test 2> Double staining of desmoglein 1 immunostaining and Fontana Masson staining in the same region of the stratum corneum was attempted.
（１）被検試料の採取
テープストリッピング法により、角層を５１歳男性の両頬から採取した。
採取した角層をスライドガラスに貼付した（図２ 参照）。
(1) Collection of test samples The stratum corneum was collected from both cheeks of a 51-year-old man by the tape stripping method.
The collected stratum corneum was attached to a glass slide (see Figure 2).
（２）第一測定工程：デスモグレイン１の免疫染色
試験１と同様の方法により、第一測定工程を行った。
(2) First measurement step: Desmoglein 1 immunostaining The first measurement step was performed in the same manner as Test 1.
（３）中間処理工程（第一測定工程後、第二測定工程前の処理）
試験１と同様の方法により、中間処理工程を行った。
(3) Intermediate treatment process (treatment after the first measurement process and before the second measurement process)
An intermediate treatment step was performed in the same manner as Test 1.
（４）第二測定工程：フォンタナマッソン染色法による組織染色
中間処理工程後の角層を、フォンタナ・アンモニア銀溶液（武藤化学株式会社 製）に、３７℃、２４時間浸漬させた。浸漬後、角層を水洗、風乾した。
風乾後の角層をＮｅｗ Ｍ・Ｘ（ポリスチレン樹脂封入剤）でスライドガラスとカバーガラスとの間に封入し、角層の観察を行った。
(4) Second measurement step: Tissue staining by Fontana Masson staining The stratum corneum after the intermediate treatment step was immersed in Fontana ammonia silver solution (manufactured by Muto Kagaku Co., Ltd.) at 37° C. for 24 hours. After soaking, the stratum corneum was washed with water and air-dried.
The air-dried stratum corneum was encapsulated between a slide glass and a cover glass using New M.X (polystyrene resin mounting medium), and the stratum corneum was observed.
（５）結果、考察
染色結果を図３、図４に示す。
なお、第一測定工程の測定結果の画像と、第二測定工程の測定結果の画像とを並列に配置し、本分野を専門とする評価者により、デスモグレイン１の存在位置と、角層におけるメラニン組織の存在位置とを対比観察した。
(5) Results and discussion The staining results are shown in Figures 3 and 4.
The image of the measurement results of the first measurement step and the image of the measurement results of the second measurement step were arranged in parallel, and an evaluator who specializes in this field determined the location of desmograin 1 and the location in the stratum corneum. Comparative observations were made of the location of melanin tissue.
図３、図４に示す通り、蛍光免疫染色によりデスモグレイン１の存在位置を測定する第一測定工程を行った後、角層におけるメラニン組織をフォンタナマッソン染色処理により測定する第二測定工程を行うことで、同一の角層におけるデスモグレイン１の存在位置と角層におけるメラニン組織の存在位置を、効率よく測定することができることがわかった。 As shown in Figures 3 and 4, after performing the first measurement step of measuring the location of desmoglein 1 by fluorescent immunostaining, the second measurement step of measuring melanin tissue in the stratum corneum by Fontana Masson staining is performed. It was thus found that the location of desmoglein 1 in the same stratum corneum and the location of melanin tissue in the same stratum corneum can be efficiently measured.
すなわち、免疫染色（Ｉｍｍｕｎｏｓｔａｉｎｉｎｇ）により特定のタンパク質の存在位置を測定する第一測定工程を行った後、角層における特定の形状的特徴の存在位置を染色処理により測定する第二測定工程を行うことで、同一の角層における特定のタンパク質の存在位置と角層内における特定組織の存在位置を対比観察することができることがわかった。 That is, after performing a first measurement step of measuring the location of a specific protein by immunostaining, a second measurement step of measuring the location of a specific shape feature in the stratum corneum by staining. We found that it is possible to compare and observe the location of specific proteins in the same stratum corneum and the location of specific tissues within the stratum corneum.
本発明は、特定のタンパク質の存在位置と角層自体の形状的特徴及び／又は角層内における特定組織の存在位置とを対比観察する技術に応用することができる。 The present invention can be applied to a technique for comparing and observing the location of a specific protein with the geometrical characteristics of the stratum corneum itself and/or the location of a specific tissue within the stratum corneum.
Claims (7)
前記第一測定工程後に、前記第一測定工程で用いた角層と同一の角層に対し、組織染色処理により、角層自体の形状的特徴及び／又は角層内における特定組織の存在位置を測定し、前記第一測定工程で取得した画像と同一の部分についての測定結果の画像を取得する第二測定工程と、
前記第一測定工程の測定結果の画像及び前記第二測定工程の測定結果の画像に基づき、同一の角層における、特定のタンパク質の存在位置と角層自体の形状的特徴及び／又は角層内における特定組織の存在位置と、を対比観察する対比観察工程と、
を有し、
前記第一測定工程と前記第二測定工程との間に、中間処理工程を有し、
前記中間処理工程は、前記第一測定工程後に２５℃以上の温水に前記角層の配置されたスライドガラスを浸し、前記角層を崩さずに前記水溶性封入剤を除くことを含む、方法。 The stratum corneum placed between a cover glass and a slide glass is encapsulated using a water-soluble mounting medium, and the location of a specific protein in the stratum corneum is measured by immunostaining , and an image of the measurement results is obtained. a first measurement step to obtain
After the first measurement step, the same stratum corneum used in the first measurement step is subjected to tissue staining to determine the geometric characteristics of the stratum corneum itself and/or the location of specific tissues within the stratum corneum. a second measurement step of measuring and obtaining an image of the measurement result for the same part as the image obtained in the first measurement step ;
Based on the image of the measurement results of the first measurement step and the image of the measurement results of the second measurement step, the position of a specific protein in the same stratum corneum, the shape characteristics of the stratum corneum itself, and/or the inside of the stratum corneum are determined. a comparative observation step of comparatively observing the location of the specific tissue in the
has
An intermediate treatment step is provided between the first measurement step and the second measurement step,
The intermediate treatment step includes immersing the glass slide on which the stratum corneum is arranged in hot water of 25° C. or higher after the first measurement step, and removing the water-soluble mounting medium without destroying the stratum corneum.
前記第一測定工程が、スライドガラスとカバーガラスとを有機溶剤に浸漬させ、前記角層を崩さずに前記油溶性接着剤を除くことを含む、請求項１～３の何れか一項に記載の方法。 When encapsulating the stratum corneum, the first measurement step includes a process of fixing a slide glass and a cover glass using an oil-soluble adhesive,
According to any one of claims 1 to 3, the first measurement step includes immersing a slide glass and a cover glass in an organic solvent and removing the oil-soluble adhesive without destroying the stratum corneum. the method of.
前記対比観察工程が、
特定のタンパク質の蛍光強度と、
特定のタンパク質の存在位置と角層自体の形状的特徴及び／又は角層内における特定組織の存在位置と、
を対比観察することを含む、請求項１～５の何れか一項に記載の方法。 The immunostaining is fluorescent staining,
The comparative observation step
Fluorescence intensity of a specific protein and
The location of a specific protein, the shape characteristics of the stratum corneum itself, and/or the location of a specific tissue within the stratum corneum,
The method according to any one of claims 1 to 5, comprising comparing and observing.
The method according to any one of claims 1 to 6, wherein the first measuring step is a step of measuring the location of desmoglein in the stratum corneum.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2019165711A JP7360283B2 (en) | 2019-09-11 | 2019-09-11 | A method for comparing and observing the location of a specific protein with the geometric characteristics of the stratum corneum itself and/or the location of a specific tissue within the stratum corneum |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2019165711A JP7360283B2 (en) | 2019-09-11 | 2019-09-11 | A method for comparing and observing the location of a specific protein with the geometric characteristics of the stratum corneum itself and/or the location of a specific tissue within the stratum corneum |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JP2021043085A JP2021043085A (en) | 2021-03-18 |
| JP7360283B2 true JP7360283B2 (en) | 2023-10-12 |
Family
ID=74863874
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2019165711A Active JP7360283B2 (en) | 2019-09-11 | 2019-09-11 | A method for comparing and observing the location of a specific protein with the geometric characteristics of the stratum corneum itself and/or the location of a specific tissue within the stratum corneum |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP7360283B2 (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2025002045A (en) * | 2023-06-21 | 2025-01-09 | 株式会社Ｓｃｒｅｅｎホールディングス | Staining condition evaluation method and computer-readable program |
| JP2025002044A (en) * | 2023-06-21 | 2025-01-09 | 株式会社Ｓｃｒｅｅｎホールディングス | Staining condition evaluation method |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2007199053A (en) | 2005-12-28 | 2007-08-09 | Kose Corp | Horny layer protein detecting method and methods for evaluating epidermis turnover and skin state using same |
| JP2016037501A (en) | 2014-08-08 | 2016-03-22 | 株式会社コーセー | Astaxanthin-containing desmoglein reducing agent |
| US20160195515A1 (en) | 2013-09-03 | 2016-07-07 | Laboratoires Expanscience | Corneodesomosomes and barrier function maturation |
| JP2017119665A (en) | 2015-08-11 | 2017-07-06 | ポーラ化成工業株式会社 | Cosmetic base for skin state improvement and screening method therefor |
-
2019
- 2019-09-11 JP JP2019165711A patent/JP7360283B2/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2007199053A (en) | 2005-12-28 | 2007-08-09 | Kose Corp | Horny layer protein detecting method and methods for evaluating epidermis turnover and skin state using same |
| US20160195515A1 (en) | 2013-09-03 | 2016-07-07 | Laboratoires Expanscience | Corneodesomosomes and barrier function maturation |
| JP2016037501A (en) | 2014-08-08 | 2016-03-22 | 株式会社コーセー | Astaxanthin-containing desmoglein reducing agent |
| JP2017119665A (en) | 2015-08-11 | 2017-07-06 | ポーラ化成工業株式会社 | Cosmetic base for skin state improvement and screening method therefor |
Non-Patent Citations (1)
| Title |
|---|
| Ueda Hiroshi，Newly devised glycerol-mounted permanent slide preparation for dissected specimens of micro-crustaceans，Plankton and Benthos Research，2018年，Vol.13 No.1 ，Page.28-31 |
Also Published As
| Publication number | Publication date |
|---|---|
| JP2021043085A (en) | 2021-03-18 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP6909227B2 (en) | Methods for swelling clinical tissue specimens | |
| US11802822B2 (en) | Multiplexed expansion (MultiExM) pathology | |
| Schiessl et al. | Long-term cell fate tracking of individual renal cells using serial intravital microscopy | |
| Cicchi et al. | Clinical nonlinear laser imaging of human skin: a review | |
| Packard et al. | Automated segmentation of light-sheet fluorescent imaging to characterize experimental doxorubicin-induced cardiac injury and repair | |
| Coffey et al. | Dynamic application of microprojection arrays to skin induces circulating protein extravasation for enhanced biomarker capture and detection | |
| JP7360283B2 (en) | A method for comparing and observing the location of a specific protein with the geometric characteristics of the stratum corneum itself and/or the location of a specific tissue within the stratum corneum | |
| JP6243123B2 (en) | Method for evaluating stratum corneum state and method for evaluating stratum corneum improvement effect of cosmetics | |
| US7632633B2 (en) | Method for determining the degree of protein oxidation in a skin sample using oxidized protein in stratum corneum as an indicator | |
| Yaroslavsky et al. | Fluorescence polarization of tetracycline derivatives as a technique for mapping nonmelanoma skin cancers | |
| Wlodek et al. | An international survey about nail histology processing techniques. | |
| Gignac et al. | Multiscale imaging of the rat brain using an integrated diceCT and histology workflow | |
| Blume-Peytavi et al. | Hair growth assessment techniques | |
| JP6419654B2 (en) | Evaluation of hair growth and scalp compressive stress | |
| JP7001600B2 (en) | Clear skin sample | |
| Presnell et al. | Full depth measurement of tenofovir transport in rectal mucosa using confocal Raman spectroscopy and optical coherence tomography | |
| CN118409096A (en) | Kit and method for detecting skin small fiber nerves | |
| Allain et al. | Multiscale characterisation of skin mechanics through in situ imaging | |
| RU2455937C1 (en) | Method of detecting pseudoexfoliation material at early stages of eye disease | |
| US20230025694A1 (en) | Silicone Hydrogel Based Fluorescent Assay and Contact Lens | |
| Fox et al. | Multimodal study of murine cardiovascular remodeling: four-dimensional ultrasound and mass spectrometry imaging | |
| CN107505293A (en) | A kind of imaging method that artery and vein vascular is separated using Two Photon Fluorescence | |
| Graham et al. | Papanicolaou smears and frozen sections on selected cutaneous neoplasms | |
| Moh et al. | Paraffin-embedded tissue (PET) blot method: application to Alzheimer disease | |
| JP2010012076A (en) | Method of measuring dermal moisture quantity |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| A621 | Written request for application examination |
Free format text: JAPANESE INTERMEDIATE CODE: A621 Effective date: 20220714 |
|
| A977 | Report on retrieval |
Free format text: JAPANESE INTERMEDIATE CODE: A971007 Effective date: 20230622 |
|
| A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20230704 |
|
| RD03 | Notification of appointment of power of attorney |
Free format text: JAPANESE INTERMEDIATE CODE: A7423 Effective date: 20230804 |
|
| A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20230818 |
|
| TRDD | Decision of grant or rejection written | ||
| A01 | Written decision to grant a patent or to grant a registration (utility model) |
Free format text: JAPANESE INTERMEDIATE CODE: A01 Effective date: 20230905 |
|
| A61 | First payment of annual fees (during grant procedure) |
Free format text: JAPANESE INTERMEDIATE CODE: A61 Effective date: 20230929 |
|
| R150 | Certificate of patent or registration of utility model |
Ref document number: 7360283 Country of ref document: JP Free format text: JAPANESE INTERMEDIATE CODE: R150 |