WO2014069519A1 - 透明化生物標本作製用キット及び透明化生物標本作製方法 - Google Patents
透明化生物標本作製用キット及び透明化生物標本作製方法 Download PDFInfo
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- WO2014069519A1 WO2014069519A1 PCT/JP2013/079388 JP2013079388W WO2014069519A1 WO 2014069519 A1 WO2014069519 A1 WO 2014069519A1 JP 2013079388 W JP2013079388 W JP 2013079388W WO 2014069519 A1 WO2014069519 A1 WO 2014069519A1
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- transparent
- nonionic surfactant
- biological specimen
- transparentized
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Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/0231—Chemically defined matrices, e.g. alginate gels, for immobilising, holding or storing cells, tissue or organs for preservation purposes; Chemically altering or fixing cells, tissue or organs, e.g. by cross-linking, for preservation purposes
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
- G01N2001/305—Fixative compositions
Definitions
- the present invention relates to a transparent biological specimen preparation kit and a transparent biological specimen preparation method. More specifically, the present invention relates to a cleared biological specimen preparation kit, a transparent biological specimen storage kit, a transparent biological specimen preparation method, and a transparent biological specimen storage method.
- the technology for making a biological specimen transparent is a technique necessary for observing a biological specimen and has been known for a long time.
- biological specimens are often observed using fluorescent dyes and are also used for observing fine tissues such as nerve cells.
- the conventional methods for clarifying biological specimens mainly have the following problems. (1) In tissue digestion with alkaline solution or proteolytic enzyme, tissue damage is likely to occur. (2) It takes a long time (several days to several months) to separate the dyeing with the alkali and glycerol mixed solution. (3) Clarification with glycerol or Scale reagent takes a long time (several days to several months). Also, it is often difficult to make transparent in specific organs such as the brain, liver and placenta. (4) The mixed solution of benzyl alcohol and benzyl benzoate is an organic solvent, and the fluorescence signal may be attenuated by treatment with the mixed solution (Non-Patent Document 3). Limited range.
- the present invention is less susceptible to tissue damage, can be transparent in a relatively short time (short period) without using an organic solvent such as benzyl alcohol or benzyl benzoate, and transparent.
- An object of the present invention is to provide a method for clarification of a biological specimen and a kit for clarification, which have a wide range of biological specimens that can be transformed. Furthermore, this invention aims at providing the preservation
- the transparent biological specimen preparation method (A) of the present invention comprises: A step (1) of obtaining a clarified transparency by treating a clarification substance as a biological specimen with a fixing solution;
- the fixing solution is (a) an aqueous solution containing formaldehyde or paraformaldehyde, a nonionic surfactant, an alkali and a buffer, or (b) an aqueous solution containing formaldehyde or paraformaldehyde, a nonionic surfactant, and an alkali.
- the concentration of the nonionic surfactant in the fixing solution is 1% or more.
- the transparent biological specimen preparation method (B) of the present invention comprises: A transparent object to be clarified is obtained by treating the clarified substance to which the biological specimen is immobilized or the clarified biological specimen preparation method (A) of the present invention with the clarification promoting liquid.
- the clarification promoting liquid is an aqueous solution containing a nonionic surfactant and an alkali, The concentration of the nonionic surfactant in the clearing promotion liquid is 1% or more.
- the transparent biological specimen preparation method (C) of the present invention comprises: The to-be-clarified material treated with the fixing liquid in the transparent biological specimen preparation method (A) of the present invention, or the transparent object treated with the fixing liquid and the clearing acceleration liquid in the transparent biological specimen preparation method (B) of the present invention.
- the preservation solution is an aqueous solution containing a nonionic surfactant and a polyhydric alcohol, and the concentration of the nonionic surfactant in the preservation solution is 1% or more.
- the cleared biological specimen preparation kit (A) of the present invention is a kit containing a fixing solution
- the fixing solution is (a) an aqueous solution containing formaldehyde or paraformaldehyde, a nonionic surfactant, an alkali and a buffer, or (b) an aqueous solution containing formaldehyde or paraformaldehyde, a nonionic surfactant, and an alkali.
- concentration of the nonionic surfactant in the fixing solution is 1% or more.
- the kit for preparing a cleared biological specimen of the present invention (B) is a kit containing a fixing solution and a clearing promoting solution
- the fixing solution is an aqueous solution containing formaldehyde or paraformaldehyde, a nonionic surfactant, an alkali and a buffer
- the clearing promoting liquid is an aqueous solution containing a nonionic surfactant and an alkali, and the concentration of the nonionic surfactant in the fixing liquid and the clearing promoting liquid is independently 1% or more. .
- the kit for the preparation of a cleared biological specimen of the present invention is a kit containing a fixing solution and a preserving solution, or a fixing solution, a clearing promoting solution and a preserving solution
- the fixing solution is an aqueous solution containing formaldehyde or paraformaldehyde, a nonionic surfactant, an alkali and a buffer
- the clearing promoting liquid is an aqueous solution containing a nonionic surfactant and an alkali
- the preserving liquid is an aqueous solution containing a nonionic surfactant and a polyhydric alcohol
- concentration of the nonionic surfactant in the fixing solution, the clarification accelerating solution and the storage solution is independently 1% or more.
- the kit for preparing a cleared biological specimen of the present invention (D) is a kit containing a clearing promoting liquid,
- the clearing promoting liquid is an aqueous solution containing a nonionic surfactant and an alkali, and the concentration of the nonionic surfactant in the clearing promoting liquid is 1% or more.
- the transparent biological specimen preparation method of the present invention has the following effects in bone staining.
- the staining time can be significantly shortened (conventional method: several days to several months, this method: one day to several days).
- Clearing time can be significantly shortened (glycerol: several days to several months, Scale: several days to several weeks or more, this method: overnight to several days)
- a large amount of bone staining can be processed (required for reproductive and developmental toxicity tests for pharmaceuticals, agricultural chemicals, etc.).
- Simple protocol that can automate bone staining.
- the transparent biological specimen preparation method of the present invention has the following effects in application examples other than bone staining. (1) Since it is made transparent in a buffer solution, it has a wide range of applications such as fluorescence observation. (2) Since it is a water-soluble preservation solution, it can be applied to whole mount immunostaining and alkaline phosphatase staining used in in situ hybridization. (3) Since the transparency is advanced so that the internal observation is possible at the fixing stage, the skeleton can be observed through the transparent skin without performing bone staining. (4) Since there is no clouding of the lens, the pathological tissue of the lens can be observed with a fixed specimen.
- the examination result about the relationship between transparency and surfactant concentration when using Triton® X100 and Tween® 20 is shown.
- Results of medaka clearing experiment are shown.
- the results of the tree frog clearing experiment are shown.
- the transparent experiment result of a mouse fetus (embryonic age 14 days) is shown.
- the transparent experiment result of a newborn (2 days after birth) is shown.
- the transparent mouse experimental result of an adult mouse is shown.
- the result of C57B6 / J female adult brain clarification experiment is shown.
- the C57B6 / J female adult brain clearing experiment results (glycerol treatment added) are shown.
- the transparent biological specimen preparation method (A) of the present invention includes at least the following step (1).
- the fixative used in step (1) is (a) formaldehyde or paraformaldehyde, a nonionic surfactant, an aqueous solution containing an alkali and a buffer, or (b) formaldehyde or paraformaldehyde, a nonionic surfactant, And an aqueous solution containing an alkali.
- the treatment with a fixative is a treatment for fixing a tissue mainly to be clarified and at the same time obtaining a gentle digestion and a remarkable clarification effect.
- Formaldehyde or paraformaldehyde is a component for tissue fixation, and each formaldehyde and paraformaldehyde can be used alone or as a mixture.
- concentration of formaldehyde or paraformaldehyde in an aqueous solution can be appropriately determined from the viewpoint that appropriate tissue fixation is possible. For example, it can be in the range of 1 to 10 w / v%, preferably 2 to It can be in the range of 7w / v%. However, it is not intended to be limited to these ranges.
- a nonionic surfactant is a component for clarifying a biological specimen, and can be used without limitation as long as it is a nonionic surfactant.
- a nonionic surfactant for example, an ester type, an ether type, an ester / ether type, and other types
- Nonionic surfactants of the type can be exemplified.
- ether type is preferable, and examples of the ether type nonionic surfactant include Triton X-100 (octylphenol poly (ethylene glycol ether) n , n is about 10. HLB 13.4 to 13.5) or Tween 20 (polysorbate 20, polyoxyethylene sorbitan monolaurate, HLB 16.7).
- Tween 20 is a kind of polysorbate, and polysorbate is a sorbitan fatty acid ester obtained by condensation of about 20 molecules of ethylene oxide.
- Tween 40 polysorbate 40, polyoxyethylene sorbitan monopalmitate, HLB15.6
- Tween60 polysorbate 60, polyoxyethylene sorbitan monostearate, HLB14.9
- Tween65 polysorbate 65, polyoxystearate polyoxyethylene
- Ethylene sorbitan HLB 10.5
- Tween 80 polysorbate 80, polyoxyethylene sorbitan oleate, HLB 15.0
- Non-ionic surfactant is a component for making a biological specimen transparent as described above. Since the biological specimen is also made clear in the fixing solution, the concentration of the nonionic surfactant in the fixing solution can be, for example, in the range of 1 to 40 v / v%. It can select suitably according to a kind and the kind of to-be-transparent thing.
- the concentration of the nonionic surfactant in the clearing promotion liquid is preferably in the range of 2 to 30% v / v%, more preferably in the range of 5 to 25% v / v%. However, it is not intended to be limited to these ranges. As a tendency, Tween 20 has a clearing effect at a lower concentration than Triton X-100.
- Tween20 is colored yellow, depending on the purpose, it may be difficult to use at a high concentration (5% or more). Concentrations in the range of 1-5 v / v% may be preferred for bright field, naked eye, and light microscopy. A combination of Triton X100 with a relatively high concentration and Tween 20 with a relatively low concentration is preferable. However, since the viscosity of TritonX100 increases rapidly when it exceeds 30%, the practical upper limit of the concentration is 25% v / v%.
- Tween 20 with an HLB of 16.7 shows a clearing effect even at a low concentration compared to Triton x-100 with an HLB of 13.4 to 5, so the nonionic surfactant HLB Therefore, there is a tendency that the transparency effect is different. Furthermore, as shown in Reference Example 1, when Tween 20 and Triton x-100 are used in combination, the transparency effect is further improved as compared with the case where they are used alone. Although the reason is not clear, in consideration of these points, the type and concentration of the nonionic surfactant can be appropriately selected within a range suitable for transparency in the fixing solution.
- Alkali is a component for biological (protein) digestion and can be a hydroxide of an alkali metal element (for example, Li, Na, K, etc.). Specifically, it can be KOH or NaOH.
- the concentration of alkali can be appropriately determined in consideration of the degree of biological (protein) digestion, and can be, for example, in the range of 0.1 ⁇ ⁇ w / v% to 10 w / v%, preferably 0.5 w / v% It is in the range of ⁇ 2 w / v%.
- Fixing solution (a) contains buffer in addition to formaldehyde or paraformaldehyde, nonionic surfactant and alkali
- fixing solution (b) contains formaldehyde or paraformaldehyde, nonionic surfactant, and Contains alkali but no buffer.
- the buffering agent is a component for adjusting the pH of the fixing solution to a predetermined range (for example, 5 to 12, preferably 7 to 11). By coexisting a buffering agent (for example, pH 5 to 8), an alkaline solution can be obtained. Tissue damage due to digestion can be further suppressed.
- the fixing solution (b) does not contain a buffering agent, the object to be clarified can be fixed by containing the above components.
- the buffer may be used alone or in combination as long as it has a pH buffering action.
- phosphate buffer e.g., phosphoric acid, sodium monohydrogen phosphate, sodium dihydrogen phosphate, potassium monohydrogen phosphate, potassium dihydrogen phosphate, sodium phosphate, potassium phosphate, etc. alone or as a mixture
- boric acid-borax buffer e.g., boric acid, sodium monohydrogen phosphate, sodium dihydrogen phosphate, potassium monohydrogen phosphate, potassium dihydrogen phosphate, sodium phosphate, potassium phosphate, etc. alone or as a mixture
- boric acid-borax buffer e buffers are preferred.
- the fixing solution (a) is, for example, 2-4% phosphate buffered formaldehyde or 2-4% phosphate buffered paraformaldehyde, an alkaline solution (1% KOH or NaOH), a nonionic surfactant ( It can be a mixed aqueous solution of 5% Triton X-100 or Tween 20).
- the fixing solution (b) is, for example, 2-4% formaldehyde aqueous solution or 2-4% paraformaldehyde aqueous solution, alkaline solution (1% KOH or NaOH), nonionic surfactant (5% Triton X -100 or Tween 20).
- the concentration of each component is not intended to be limited to the above value, but is merely an example.
- the treatment with the fixing solution of the object to be clarified is performed, for example, with a predetermined amount (at least the amount of the object to be clarified sinks in the fixing solution) of room temperature (for example, 5 to 35 ° C., preferably 15 to 30 ° C., and so on) ) Or at a temperature in the range of room temperature to 60 ° C., by immersing the material to be transparent in the range of 1 to 48 hours, preferably in the range of 1 to 24 hours, more preferably in the range of 2 to 12 hours. If the temperature is higher, the fixation tends to be completed in a short time, but depending on the type of the object to be translucent, there is a risk of damage to the tissue. It is preferable to adjust.
- room temperature for example, 5 to 35 ° C., preferably 15 to 30 ° C., and so on
- the temperature and time are merely examples, and are not intended to be limited to these ranges.
- treatment at a relatively low temperature such as room temperature is preferable from the viewpoint of suppressing tissue damage due to alkaline digestion.
- the treatment with the fixing solution of the object to be clarified can be performed while irradiating microwaves in a part or all of the treatment operations.
- the microwave can have a frequency of 2.45 GHz, for example, but is not intended to be limited thereto.
- Transparency can be further promoted by performing the treatment with the fixing solution of the object to be transparent while irradiating the microwave. Note that the output of the microwave to be irradiated can be appropriately determined in consideration of the type and size of the object to be transparent, the amount of the fixing liquid, and the like.
- the biological specimen can be, for example, fish, amphibians, reptiles, or mammals (however, in the case of the whole living body, excluding humans).
- the biological specimen can be a living tissue, and can be any tissue of the living body, for example, a viscera, blood vessel, nerve, brain, or bone.
- transparent specimens of human organs and the like can also be technically prepared.
- the preparation is performed with ethical considerations, such as obtaining approval from the ethics committee.
- these are merely examples and are not limited as long as they are biological specimens.
- the object to be clarified can be fish, amphibians, birds, mammals or tissues thereof.
- the biological specimen that is the object to be clarified can be one that has been subjected to a staining treatment or the like in advance.
- the dye used for the dyeing process is not particularly limited, and may be pigment ink or the like, or dyeing with a fluorescent dye.
- the object to be clarified can be washed if necessary.
- distilled water, buffer solution, ethylene glycol / PB (S) or the like can be used. Washing can be performed at room temperature.
- the object to be clarified can be bleached, degreased or bleached and degreased as necessary after treatment with a fixing solution, or treatment with a fixing solution and washing.
- bleaching for example, hydrogen peroxide can be used.
- degreasing for example, acetone or ethanol can be used.
- a transparent object to be transparent is obtained by treatment with a fixing solution of the object to be transparent.
- the transparent biological specimen obtained can be used as it is for the next observation, for example.
- the transparency may be insufficient depending on the type of the object to be transparentized.
- Such a material to be clarified can be further subjected to the transparent biological specimen preparation method (B) of the present invention or the transparent biological specimen preparation method (C) of the present invention to further promote transparency.
- the transparent biological specimen preparation method (B) of the present invention is a transparentized specimen prepared by immobilizing a biological specimen by a method other than the transparent biological specimen preparation method (A) of the present invention, or the transparent biological specimen preparation method of the present invention.
- (A) includes a step (2) of treating the object to be clarified treated with the fixing solution with a clarification accelerating solution.
- the clearing promoting liquid used in the step (2) is an aqueous solution containing a nonionic surfactant and an alkali.
- Nonionic surfactants and alkalis can be the same as those used in the fixing solution.
- the nonionic surfactant is a component for clarifying the biological specimen, and further stabilizes the biological specimen after immobilization with the clarification promoting liquid.
- the concentration of the nonionic surfactant in the clearing promotion liquid can be, for example, in the range of 1 to 40% w / v%, and the type of nonionic surfactant, the type of the object to be clarified, and the fixing solution Can be appropriately selected according to the treatment conditions (including the composition of the fixing solution).
- the concentration of the nonionic surfactant in the clearing promoting liquid is preferably in the range of 2 to 20% w / v%, more preferably in the range of 5 to 10% w / v%. However, it is not intended to be limited to these ranges.
- Alkali is a component for digestion of living body (protein), and its concentration can be appropriately determined in consideration of the degree of digestion of living body (protein) as in the case of the fixative.
- 0.1 w / v% It can be in the range of ⁇ 10 w / v%, and is preferably in the range of 0.5 / w / v% to 2 / w / v%.
- the clearing solution does not need to contain a buffering agent (it may contain a buffering agent if necessary), and the digestive action of the living body (protein) by alkali is stronger than the fixing solution, but the fixing solution Thus, mild digestion has already been performed, and even when a clarification accelerating solution that does not contain a buffering agent is used, tissue damage does not become significant. However, it is preferable to adjust the alkali concentration according to the type of tissue to promote transparency while suppressing tissue damage.
- the object to be clarified is the object to be clarified treated with the fixing liquid in the method for preparing a clarified biological specimen (A) of the present invention, or the biological specimen is immobilized by a method other than the method for preparing a clarified biological specimen (A) of the present invention. It is a transparent object.
- the method other than the transparent biological specimen preparation method (A) of the present invention can be a conventionally known method.
- the biological specimen contains formaldehyde or paraformaldehyde, and includes a nonionic surfactant and an alkali. It can be a method of treating with an aqueous solution not containing one or both.
- Aqueous solutions containing formaldehyde or paraformaldehyde and not containing one or both of nonionic surfactants and alkalis are, for example, 2-4% formaldehyde aqueous solution, 2-4% paraformaldehyde aqueous solution, 2-4% phosphoric acid. It can be buffered formaldehyde or 2-4% phosphate buffered paraformaldehyde.
- the treatment of the object to be clarified with the clarification accelerating liquid is carried out, for example, by adding a predetermined amount (at least the amount of the entire clarified object to sink in the clarification accelerating liquid) to room temperature (for example, 5 to 35 ° C., preferably 15 To be clarified at a temperature ranging from room temperature to 60 ° C. for 10 minutes to 48 hours, preferably 30 minutes to 24 hours, more preferably 1 hour to 12 hours. Is performed by dipping. The higher the temperature, the more likely it is that the effect of promoting transparency will be obtained in a short time.However, depending on the type of the material to be transparentized, there is a risk that damage to the tissue may increase. It is preferable to adjust appropriately. However, the temperature and time are merely examples, and are not intended to be limited to these ranges.
- the treatment of the object to be clarified with the clarification accelerating liquid can be performed while irradiating microwaves in a part or all of the treatment operations.
- the microwave can have a frequency of 2.45 GHz, for example, but is not intended to be limited thereto.
- the transparency can be further promoted, and the transparency can be achieved in a shorter time.
- the specimen is damaged in a relatively short time of about 10 to 90 minutes by performing the treatment with the above-described transparency promoting liquid while irradiating microwaves at a temperature of 40 to 50 ° C. Transparency is possible without any problem.
- the output of the microwave to be irradiated can be appropriately determined in consideration of the type and size of the object to be transparent, the amount of the transparentization promoting liquid, and the like.
- a transparent biological specimen can be obtained by treating the object to be clarified with a clarification promoting liquid.
- the transparent biological specimen obtained can be used as it is for the next observation, for example.
- the transparency may be insufficient depending on the type of the object to be transparentized.
- Such a transparent substance can be further subjected to the transparent biological specimen preparation method (C) of the present invention to further promote transparency. Even when storage is necessary, it is preferable to store by applying the transparent biological specimen preparation method (C) of the present invention.
- the transparent biological specimen clearing method (C) of the present invention is the transparentized specimen prepared in the transparent biological specimen preparing method (A) of the present invention (A) or the transparent biological specimen preparing method of the present invention (B). ), A step (3) of obtaining the transparentized product by treating or storing the transparentized product treated with the fixing solution and the clearing promoting solution with a storage solution.
- the preservation solution used in step (3) is an aqueous solution containing a nonionic surfactant and a polyhydric alcohol, and the concentration of the nonionic surfactant is 1% or more.
- the nonionic surfactant is a component for making the biological specimen transparent, and is a component for maintaining the transparency of the transparent biological specimen.
- the nonionic surfactant those similar to those used in the fixing solution and the clarification accelerating solution can be used.
- the concentration of the nonionic surfactant in the preservation solution can be, for example, in the range of 1 to 40%.
- the clarification may further proceed and the transparency is maintained.
- the non-ionic surfactant HLB tends to have a difference in the clarification promoting effect and the transparency maintaining effect.
- Polyhydric alcohol is a component for promoting transparency, and examples include glycol compounds, and examples of glycol compounds include ethylene glycol.
- the concentration of the glycol compound can be appropriately determined in consideration of the transparency of the sample. For example, it can be in the range of 1 to 50% w / v%, preferably in the range of 10 to 30% w / v%. be able to. However, it is not intended to be limited to these ranges.
- the preservation solution may contain dimethyl sulfoxide (DMSO) in addition to the nonionic surfactant and the polyhydric alcohol.
- DMSO dimethyl sulfoxide
- the content of DMSO can be, for example, in the range of 0.5 to 10% v / v%, and the DMSO addition effect varies depending on the type of tissue or organ to be treated. In the case of general organs, a range of 0.5 to 10% v / v% is appropriate. In the case of the brain, the addition of DMSO may cause swelling, so a relatively small range of 0.5 to 1% v / v% is appropriate for long-term storage. For short-term observations, DMSO can be used up to 10% v / v%.
- the treatment with the preservation solution of the object to be clarified treated with the fixing solution and / or the clarification accelerating solution is performed, for example, in a predetermined amount (at least the amount of the whole object to be clarified sinks in the preservation solution) of room temperature (for example, 5 ⁇ 35 ° C, preferably 15-30 ° C, and so on) or at a temperature ranging from room temperature to 60 ° C, 10 minutes to 48 hours, preferably 30 minutes to 24 hours, more preferably 1 hour to 12 hours. It is performed by immersing the object to be transparent in the range of.
- the temperature and time are merely examples, and are not intended to be limited to these ranges.
- it can preserve
- the treatment of the object to be clarified with the preservation solution can be performed while irradiating with microwaves in a part or all of the treatment operations.
- the microwave can have a frequency of 2.45 GHz, for example, but is not intended to be limited thereto.
- the transparency can be further promoted, and the transparency can be achieved in a shorter time.
- the specimen is not damaged in a relatively short time of about 10 to 90 minutes. Transparency is possible.
- the output of the microwave to be irradiated can be appropriately determined in consideration of the type and size of the object to be transparent, the amount of the preservation solution, and the like.
- a transparent biological specimen can be obtained by treating the object to be clarified with a fixative.
- the transparent biological specimen obtained can be used as it is for the next observation, for example, or can be stored as it is.
- the transparency may be insufficient depending on the type of the object to be transparentized.
- transparency can be further promoted by repeating and implementing a process (2) and a process (3) again. Or when transparency is inadequate, it can also attach
- the treatment using the transparent additional accelerating liquid can be performed on the transparent biological specimen obtained by the transparent biological specimen transparentizing method (A) or (B) of the present invention.
- the transparent biological specimen obtained by the transparent biological specimen transparent method (A) to (C) of the present invention is immersed in a transparent additional acceleration solution composed of a trihydric alcohol solution or a trihydric alcohol-containing aqueous solution, and then It is also possible to obtain a transparent object to be transparent by holding the object to be transparent obtained after dipping in the transparent additional promoting liquid in the storage solution.
- the trihydric alcohol is a component that further promotes transparency, and in some cases, the transparency can be further promoted by this treatment. Examples of the trihydric alcohol include glycerol.
- the transparent additional promoting liquid treatment is effective, for example, for increasing the transparency of bones and nerve fibers.
- the treatment with the clearing additional acceleration liquid of the object to be clarified treated with the fixing liquid, the clarification accelerating liquid or the storage liquid is, for example, storing a predetermined amount (at least the entire amount of the clarified object sinks in the clarification additional accelerating liquid) In the liquid at room temperature (for example, 5 to 35 ° C., preferably 15 to 30 ° C., the same applies below) or at a temperature in the range of room temperature to 60 ° C., a range of 30 minutes to 48 hours, preferably a range of 1 hour to 24 hours, Preferably, it is carried out by immersing the object to be transparent in the range of 1 to 12 hours.
- the temperature and time are merely examples, and are not intended to be limited to these ranges.
- the treatment of the object to be clarified with the clarification additional accelerating liquid can be performed while irradiating microwaves in a part or all of the processing operations.
- the microwave can have a frequency of 2.45 GHz, for example, but is not intended to be limited thereto.
- the transparentization can be further promoted, and the transparentization can be performed in a shorter time.
- the specimen can be damaged in a relatively short time of about 10 to 90 minutes by performing the treatment with the above-mentioned transparent additional promoting liquid while irradiating microwaves at a temperature of 40 to 50 ° C. Transparency is possible without giving.
- the output of the microwave to be irradiated can be appropriately determined in consideration of the type and size of the object to be transparent, the amount of the transparent additional promoting liquid, and the like.
- step IV 1-4 The basic protocol (step IV 1-4) for tissue clearing that can be used in the above (1) fixative treatment, (2) clearing promotion liquid treatment, and (3) preservation liquid treatment is exemplified below.
- Step 1 Fixing solution for preparing transparent specimen: room temperature, several hours to several days (a) 2-4% phosphate buffered formaldehyde or 2-4% phosphate buffered paraformaldehyde, 5% Triton X-100 or 5% Tween20, 1% KOH mixture, (b) 2-4% formaldehyde aqueous solution or 2-4% paraformaldehyde aqueous solution, 5% Triton X-100 or 5% Tween20, 1% KOH mixture
- Step 2 Cleaning: 30 minutes at room temperature-overnight (i) distilled water, or (ii) Tris-HCl buffer (TB (S), pH 7.5), or (iii) phosphate buffer (PB (S), pH 7.2-7.4), or (iv) 40% ethylene glycol / PB (S)
- Bleaching (i) 0.3% hydrogen peroxide, 50% (70-50%) acetone, 50% (30-50%) ethylene glycol mixture, or (ii) 0.3% hydrogen peroxide solution, 50% ethanol, 50% ethylene glycol mixed solution, room temperature, several hours to overnight
- Degreasing (i) 50% (70-50%) acetone, 50% (30-50%) ethylene glycol mixture, or (ii) 50% ethanol, 50% ethylene glycol mixture, room temperature, several hours to several days, or (iii) 50% ethanol, 50% glycerol mixed solution * The degreasing action is superior to the acetone mixed solution of (i). * For adult mouse brains, 1 day of bleaching and 1 day -2 days of degreasing are appropriate. In the adult rat brain, 1 day of bleaching and 2 to 3 days of defatting are appropriate.
- Step 3 Tissue clearing promoting solution: room temperature or 50 ° C, 30 minutes to 2 days Clearing promoting solution 1: 20% (5-20%) Triton X-100, 1% KOH, or clearing promoting solution 2:20 % (5-20%) Tween20, 1% KOH, or Clarification Solution 3: 10% (5-20%) Triton X-100, 10% (5-25%) Tween20, 1% KOH Transparency promoting effect: Accelerating liquid 3> Accelerating liquid 1> Accelerating liquid 2
- Step 4 Clear specimen storage solution: room temperature or 50 ° C, 30 minutes to 2 days (clearing is accelerated at 50-60 ° C)
- Stock solution 1 20% (5-20%) Triton X-100, 20% ethylene glycol, or stock solution 2: 20% (5-20%) Tween 20, 20% ethylene glycol, or stock solution 3: 25% ( 5-25%) Triton X-100, 5% (5-1%) Tween20, 20% Ethylene glycol stock solution 4: 20% (5-20%) Triton X-100, 20% ethylene glycol, 10% (0.5 -10%) DMSO (dimethyl sulfoxide) (pH 8-9) Clarification promoting effect: preservation solution 4> preservation solution 3> preservation solution 1> preservation solution 2
- Step IV4 If the transparency of the specimen obtained in Step IV4 is insufficient, the transparency can be further improved by repeating Step III-3 clarification promoting solution and Step IV4 storage solution.
- any one of the transparent specimen storage solutions 1-4 can be used for storage (transparency: 4> 3> 1> 2).
- Step 5 When it is desired to further increase the transparency of the specimen, or when it is desired to increase the transparency of bones, nerve fibers, and the like, the following transparent additional promoting liquid treatment can be added.
- the clearing booster solution can be a solution of 100% glycerol or an aqueous solution of 50% or more of glycerol. The higher the glycerol concentration, the higher the effect of promoting transparency.
- a specific protocol is, for example, as follows.
- Emulsification Incubate with 100% glycerol for 30 minutes to several hours at room temperature. As soon as the specimen is added, emulsification begins and the solution becomes milky white. At the same time, the transparency of the specimen advances rapidly and becomes harder. In the clear specimen preservation solution in Step 4, tissue swelling is observed, but this glycerol treatment contracts.
- the soft tissue undergoes a treatment with the transparent additional accelerating liquid, and the transparency of the soft tissue is drastically increased.
- the treatment with the clearing additional promoting solution is particularly suitable for clearing the brain.
- milky white precipitate may occur in white matter fibers (the same applies to the ossification site).
- heating at 50 ° C-60 ° C is effective.
- the brain may suddenly melt (disintegrate) and become liquefied. It is preferable to carry out.
- the color tone of the ossification site emphasized in the fetal white color also drops (transparent). Therefore, when it is better not to see the bone, the treatment with the transparent additional promoting liquid is effective.
- the kit for preparing a cleared biological specimen of the present invention comprises a kit (A) fixing solution, a kit (B) fixing solution and a clearing promoting solution, a kit (C) fixing solution and a preserving solution, or a fixing solution, a clearing promoting solution and Each contains a preservative solution and a kit (D) clearing promoting solution.
- the fixing solution is (a) an aqueous solution containing formaldehyde or paraformaldehyde, a nonionic surfactant, an alkali and a buffer, or (b) an aqueous solution containing formaldehyde or paraformaldehyde, a nonionic surfactant, and an alkali. It is. Fixing solution (b) does not contain a buffer.
- the clearing promotion liquid is an aqueous solution containing a nonionic surfactant and an alkali.
- the preservation solution is an aqueous solution containing a nonionic surfactant and a polyhydric alcohol.
- the concentration of the nonionic surfactant in the fixing solution, the clarification accelerating solution, and the preservation solution is independently 1% or more.
- the composition, concentration, and the like of the fixing solution, the clearing promoting solution, and the preserving solution are the same as those described in the transparent biological specimen preparation methods (A), (B), and (C).
- the kit of the present invention is appropriately used in the transparent biological specimen preparation methods (A) to (C) of the present invention.
- the kit of the present invention may further include a clearing additional acceleration liquid composed of a trihydric alcohol solution or a trihydric alcohol-containing aqueous solution.
- the trihydric alcohol is glycerol, for example.
- the composition, concentration, and the like of the clearing additional acceleration liquid are the same as those described in the transparent biological specimen preparation method and the clearing additional acceleration liquid treatment.
- Reference example 1 Examination of composition of clear specimen preservation solution (1) Examination of Triton X100 and Tween20 concentrations The liver of an adult rat with pigment ink injected into the blood vessel was used. After perfusion with 2% phosphate buffered formaldehyde (paraformaldehyde), 5% Triton X-100, and 1% KOH, and fixed by immersion for 12 hours in the same fixative, 40% ethylene glycol / PB (phosphate) Buffer solution) at room temperature.
- Triton X-100 (Tx100): 20%, 10%, 5%, 1.25%
- Tween20 40%, 20%, 10%, 5%, 1.25%
- Tx + Tw (same concentration): 20%, 10%, 5%, 1.25% Does not include Tx-Tw: 0%
- Triton 100 was not examined because it solidifies at room temperature. -The effect of transparency was confirmed in the treatment group on the left side of the red line. ⁇ Triton X-100 and Tween 20 alone showed a clearing action, but the mixed solution of both showed clearing action from a lower concentration range.
- Example 1 Transparency of medaka and tree frog ⁇ Conditions> (1) Fixed 2% phosphate buffered formaldehyde (paraformaldehyde), 5% Triton X-100, 1% KOH mixture at room temperature, 1 hour to overnight
- Tissue clearing liquid promotion promoting liquid 2 20% Tween20, 1% KOH Room temperature, 10-30 minutes
- the transparency is high, and the soft tissue is so transparent that it is difficult to recognize with the naked eye.
- the soft tissue becomes transparent, the very early ossification site is dyed in white (presumed to be the effect of Tween20), so it can be used as a very sensitive bone staining method without special bone staining. Can be applied (the cartilage is not stained).
- the placenta is also transparent.
- Example 3 Clarification of adult mouse digestive tract tissue (ink injection from artery) ⁇ Conditions> (1) Fixed 2% phosphate buffered formaldehyde (paraformaldehyde), 5% Triton X-100, 1% KOH mixture, Room temperature, 1 hour to overnight
- Example 4 Clarification of adult mouse brain (part 1) ⁇ Conditions> (1) Fixed 2% phosphate buffered formaldehyde (paraformaldehyde), 5% Triton X-100, 1% KOH mixture, Room temperature, 1 hour to overnight
- FIG. 8 shows C57B6 / J adult female brain fixed with 2% phosphate buffered formaldehyde, 5% Triton X-100, 1% KOH mixed solution (room temperature, overnight), and then with acetone / ethylene glycol mixed solution
- FIG. 8B shows the result of treatment with the accelerating solution 3 (room temperature, 12 hours) and the storage solution 3 (room temperature, 24 hours).
- the gray matter was very transparent, but a milky white precipitate was formed in the white matter (nerve fibers).
- the component of the milky white precipitate formed in the white matter is unknown, but it was possible to remove the precipitate by incubating the solution at 50 ° C. for 12 to 48 hours.
- the results are shown in FIG.
- the white precipitate almost disappeared, but some turbidity was observed in the white fibers in the dark field observation. From the surface of the dorsal cerebral cortex, blood vessels running on the surface of the brain base can be observed under a microscope.
- Example 5 ICR mouse adult (9 wks, cocoon) brain transparency (part 2) ⁇ Conditions> (1) Fixed 2% PFA (paraformaldehyde), 5% Triton X100, 0.5% KOH, stored in fixative for several months until use (2) Pretreatment (washing) 20% ethylene glycol, 5% Triton X-100, 1% KOH, room temperature, overnight (3) Stock solution (clearing treatment 1) 20% ethylene glycol, 25% Triton X-100, pH 8.5, room temperature, overnight (several hours are enough) (4) Preservation solution (clarification treatment 4) 20% ethylene glycol, 20% Triton X-100, 10% DMSO, pH 8.5, room temperature, overnight (several hours are enough)
- FIG. 10 shows the results (photographs) after the treatments (2), (3), and (4).
- Example 5 does not require the use of glycerol and heating in Example 4.
- the transparency proceeds, but the transparency is almost completed after the processing of (3).
- the treatment (4) the transparency becomes more difficult as it is difficult to see.
- the preservation solution (4) contains 10% DMSO
- the specimen expands and is suitable for short-time observation.
- the example of the brain is shown in the present embodiment, it has been confirmed that the transparency is similarly progressed in the lung. In the case of the lung, the specimen does not swell even if the preservation solution of (4) contains 10% DMSO.
- Example 6 Rat adult forearm (ink injection into artery) The specimen preparation process is in accordance with the brain specimen preparation process described in Example 4. The results are shown in FIG. -Clarified with all muscles left. Bones can be clearly seen through the thick muscles of the forearm. ⁇ The vascular network distributed in the muscle can be seen through. ⁇ Muscle fiber running on the focus plane of the microscope can be confirmed (observation like tomography is possible).
- Example 7 Rapid bone staining method ⁇ Conditions> (1) Fixed: Overnight to several days, room temperature 2% phosphate buffered formaldehyde (paraformaldehyde), 5% Triton X-100, 1% KOH
- Clarification promotion and washing 50 ° C, several hours to overnight Clarification promotion liquid 2: 20% Tween20, 1% KOH
- Clarification accelerating solution 1 20% Triton X-100, 1% KOH, 50 °C Clarification promoting solution 3: 10% Triton X-100, 10% Tween20, 1% KOH
- ⁇ Clearing promotion liquid 2 is suitable for fish and frogs.
- Clarification Acceleration Solution 3 is good. • If the specimen is translucent when fixed, the staining process will be completed in 3-4 hours. -Although it can be washed at room temperature, it is slow and preferably 50 ° C. ⁇ In neutral to acidic environments, rapid tissue destruction occurs, so add KOH to the clearing solution.
- FIG. 12 and 13 show the obtained bone staining specimen of fish (fixed: 2% phosphate buffered paraformaldehyde, 5% Triton X-100, 1% KOH).
- the medaka shown in FIG. 12 is completed in half a day.
- Transparency is higher than glycerol specimens.
- Even a relatively large specimen is completed in about 24 hours.
- Example 8 (Method Using Fixation Solution not Containing Phosphate Buffer Solution) Condition 1: Immobilization: Fix with fixative (2% paraformaldehyde, 5% Triton X-100, 1% KOH mixture) (room temperature, overnight) Storage: Store in storage solution (25% Triton X100, 1% Tween 20, 20% polyethylene glycol)
- Condition 3 transparent treatment of the present invention after normal fixation: Immobilization: Fix with fixative (4% paraformaldehyde) (room temperature, overnight) Transparency Acceleration: Fix with transparency accelerating solution (5% Triton X-100, 1% KOH mixture) (room temperature, overnight) Storage: Store in stock solution (25% Triton X100, 1% Tween 20, 20% polyethylene glycol)
- FIG. 14 shows photographs of the specimen after fixation and the specimen after clarification and storage.
- the number in the photo is the condition number.
- Example 9 (Bone staining method using a fixative containing no phosphate buffer) Condition 1: Immobilization: Fix with fixative (2% paraformaldehyde, 5% Triton X-100, 1% KOH mixture) (room temperature, overnight) Staining: Bone staining with Alizarin red S Acceleration: Transparency accelerating solution (20% Tween 20, 1% KOH), (42 °C, overnight) Transparent additional acceleration: Transparent additional acceleration liquid (glycerol (100%))
- FIG. 1 A photograph of the specimen after immobilization and the specimen after clarification and storage is shown in FIG. The number in the photo is the condition number.
- the present invention is useful in fields that require observation of biological specimens.
Abstract
Description
本発明は、透明化生物標本作製用キット及び透明化生物標本作製方法に関する。より詳細には、透明化生物標本作製用キット及び透明化した生物標本の保存用キット、並びに透明化生物標本作製方法及び透明化した生物標本の保存方法に関する。
(1)アルカリ溶液またはタンパク分解酵素による組織消化では、組織の破損が生じやすい。
(2)アルカリ及びグリセロール混合液による染色の分別には長時間(数日から数ヶ月)を要する。
(3)グリセロールまたはScale試薬による透明化には長時間(数日から数ヶ月)を要する。また、脳、肝臓や胎盤など、特定の臓器では透明化が困難なことが多い。
(4)ベンジルアルコール及び安息香酸ベンジル混合液は有機溶媒であり、同混合液による処理によって蛍光シグナルが減弱することがあるなど(非特許文献3)、透明化後の観察を含めて、応用の範囲が限られる。
生物標本である被透明化物を固定液で処理して、透明化した被透明化物を得る工程(1)を含み、
前記固定液は、(a)ホルムアルデヒドまたはパラホルムアルデヒド、非イオン系界面活性剤、アルカリ及び緩衝剤を含有する水溶液または(b) ホルムアルデヒドまたはパラホルムアルデヒド、非イオン系界面活性剤、及びアルカリを含有する水溶液であり、
前記固定液における非イオン系界面活性剤の濃度は、1%以上である。
生物標本を固定化した被透明化物または前記本発明の透明化生物標本作製方法(A)において固定液で処理した被透明化物を透明化促進液で処理して、透明化した被透明化物を得る工程(2)を含み、
前記透明化促進液は、非イオン系界面活性剤及びアルカリを含有する水溶液であり、
前記透明化促進液における非イオン系界面活性剤の濃度は、1%以上である。
前記本発明の透明化生物標本作製方法(A)において固定液で処理した被透明化物、または前記本発明の透明化生物標本作製方法(B)において固定液及び透明化促進液で処理した被透明化物を保存液で処理または保存して、透明化した被透明化物を得る工程(3)を含み、
前記保存液は、非イオン系界面活性剤及び多価アルコールを含有する水溶液であって、前記保存液における非イオン系界面活性剤の濃度は、1%以上である。
前記固定液は、(a)ホルムアルデヒドまたはパラホルムアルデヒド、非イオン系界面活性剤、アルカリ及び緩衝剤を含有する水溶液または(b) ホルムアルデヒドまたはパラホルムアルデヒド、非イオン系界面活性剤、及びアルカリを含有する水溶液であり、前記固定液における非イオン系界面活性剤の濃度は、1%以上である。
前記固定液は、ホルムアルデヒドまたはパラホルムアルデヒド、非イオン系界面活性剤、アルカリ及び緩衝剤を含有する水溶液であり、
前記透明化促進液は、非イオン系界面活性剤及びアルカリを含有する水溶液であり、かつ
前記固定液及び透明化促進液における非イオン系界面活性剤の濃度は、独立に、1%以上である。
前記固定液は、ホルムアルデヒドまたはパラホルムアルデヒド、非イオン系界面活性剤、アルカリ及び緩衝剤を含有する水溶液であり、
前記透明化促進液は、非イオン系界面活性剤及びアルカリを含有する水溶液であり、かつ
前記保存液は、非イオン系界面活性剤及び多価アルコールを含有する水溶液であって、
前記固定液、透明化促進液及び保存液における非イオン系界面活性剤の濃度は、独立に、1%以上である。
前記透明化促進液は、非イオン系界面活性剤及びアルカリを含有する水溶液であり、かつ
前記透明化促進液における非イオン系界面活性剤の濃度は、1%以上である。
(1)染色の時間を著しく短縮できる(従来法:数日~数カ月,本法:1日から数日)。
(2)透明化の時間を著しく短縮できる(グリセロール:数日~数カ月,Scale:数日~数週間以上、本法:一晩~数日)
(3)大量の骨染色を処理することが可能(医薬品や農薬等の生殖発生毒性試験で必須)。
(4)シンプルなプロトコルであり、骨染色の自動化が可能。
(1)緩衝液中で透明化されているため、蛍光観察など応用範囲が広い。
(2)水溶性の保存液であるため、ホールマウント免疫染色やin situ hybridizationで用いられるアルカリフォスファターゼ染色に応用可能である。
(3)固定の段階で内部観察が可能なほど透明化が進むため、骨染色を施さなくても、透明化した皮膚を通して骨格の観察をすることができる。
(4)水晶体の白濁がないため、水晶体の病理組織を固定標本で観察可能である。
(5)固定液の界面活性剤にTween 20を用いた場合、メラニン色素が残る為、透明化された臓器組織中に広がるメラノサイトの分布を広範囲で観察することができる。
(6)従来法では透明化が困難であった脳、肝臓、腎臓、胎盤についても、本法により(主に固定液の組成を本発明の組成にすることで)短時間で十分透明化が得られる。
(7)厚切り切片(40μm以上)の透徹に本法を応用することで、顕微鏡観察可能な深さが飛躍的に伸びる(組織標本のマウント剤としての応用が可能)。
本発明の透明化生物標本作製方法(A)は、少なくとも以下の工程(1)を含む。
生物標本である被透明化物を固定液で処理する工程(1)
工程(1)で使用する固定液は、(a)ホルムアルデヒドまたはパラホルムアルデヒド、非イオン系界面活性剤、アルカリ及び緩衝剤を含有する水溶液または(b) ホルムアルデヒドまたはパラホルムアルデヒド、非イオン系界面活性剤、及びアルカリを含有する水溶液である。固定液での処理は、被透明化物を主に、組織を固定すると同時に、穏やかな消化と著しい透明化作用を得るための処理である。
但し、各成分の濃度は、上記値に限定される意図ではなく、あくまでも例示である。
本発明の透明化生物標本作製方法(B)は、本発明の透明化生物標本作製方法(A)以外の方法で生物標本を固定化した被透明化物、または本発明の透明化生物標本作製方法(A)において固定液で処理した被透明化物を透明化促進液で処理する工程(2)を含む。
工程(2)で使用する透明化促進液は、非イオン系界面活性剤及びアルカリを含有する水溶液である。非イオン系界面活性剤及びアルカリは、固定液で用いたものと同様のものを利用できる。
本発明の透明化生物標本透明化方法(C)は、本発明の透明化生物標本作製方法(A)において固定液で処理した被透明化物、または前記本発明の透明化生物標本作製方法(B)において固定液及び透明化促進液で処理した被透明化物を保存液で処理または保存して、透明化した被透明化物を得る工程(3)を含む。
工程(3)で使用する保存液は、非イオン系界面活性剤及び多価アルコールを含有する水溶液であって、前記非イオン系界面活性剤の濃度は、1%以上である。非イオン系界面活性剤は、生物標本を透明化するための成分であり、透明化された生物標本の透明性を維持する成分である。非イオン系界面活性剤は、固定液及び透明化促進液で用いたものと同様のものを利用できる。
本発明の透明化生物標本透明化方法(A)~(C)で得られた透明化生物標本は、3価アルコール溶液または3価アルコール含有水溶液からなる透明化追加促進液に浸漬し、次いで前記透明化追加促進液に浸漬した後に得られる被透明化物を、前記保存液に保持して、透明化した被透明化物を得ることもできる。3価アルコールは、透明化をより促進する成分であり、この処理により、透明化をより促進することができる場合がある。3価アルコールとしては、例えば、グリセロールを挙げることかできる。透明化追加促進液処理は、例えば、骨、神経線維の透明度を上げる場合に有効である。
(a)2-4% リン酸緩衝ホルムアルデヒドまたは2-4% リン酸緩衝パラホルムアルデヒド、5% Triton X-100または5% Tween20、1% KOH混合液、
(b)2-4%ホルムアルデヒド水溶液または2-4%パラホルムアルデヒド水溶液、5% Triton X-100または5% Tween20、1% KOH混合液
(i)蒸留水、または
(ii)Tris-HCl緩衝液(TB(S), pH7.5)、または
(iii)リン酸緩衝液(PB(S), pH7.2-7.4)、または
(iv)40% エチレングリコール/PB(S)
漂白:(i)0.3%過酸化水素水、50%(70-50%)アセトン、50%(30-50%)エチレングリコール混合液、または
(ii)0.3%過酸化水素水、50%エタノール、50%エチレングリコール混合液、室温、数時間~一晩
(ii)50% エタノール、50% エチレングリコール混合液、室温、数時間~数日、または
(iii)50% エタノール、50% グリセロール混合液
*脱脂作用は(i)のアセトン混合液が優れる。
*マウス成獣脳では漂白1日、脱脂1日-2日が適当である。
ラット成獣脳では漂白1日、脱脂2日-3日が適当である。
透明化促進液1:20%(5-20%)Triton X-100、1% KOH、または
透明化促進液2:20%(5-20%)Tween20、1% KOH、または
透明化促進液3:10%(5-20%)Triton X-100、10%(5-25%)Tween20、1% KOH
透明化促進効果:促進液3>促進液1>促進液2
保存液1:20%(5-20%)Triton X-100、20% エチレングリコール、または
保存液2:20%(5-20%)Tween20、20% エチレングリコール、または
保存液3:25%(5-25%)Triton X-100、5%(5-1%)Tween20、20% エチレングリコール
保存液4: 20%(5-20%)Triton X-100、20% エチレングリコール、10% (0.5-10%)DMSO(ジメチルスルホキシド)(pH 8-9)
透明化促進効果:保存液4>保存液3>保存液1>保存液2
標本の透明度を更に上げたい場合、または骨、神経線維等の透明度を上げたい場合には、以下に示す透明化追加促進液処理を追加することができる。透明化追加促進液は、グリセロール100%の溶液であるか、またはグリセロール50%以上の水溶液であることができる。グリセロール濃度が高いほど、透明化追加促進効果は高くなる。具体的なプロトコルは例えば、以下の通りである。
標本を入れると同時に、乳化が始まり、溶液は乳白色となる。同時に、標本の透明化は急激に進み、硬くなる。Step 4の透明標本保存液では組織膨化が認められるが、本グリセロール処理では収縮する。
・透明化追加促進液による処理は、特に脳の透明化に適した処理である。
・Step 1-4の処理では、白質の線維に乳白色の沈殿が生じる(骨化部位も同様)場合がある。これを取り除く処理として、50℃-60℃の加熱が有効であるが、72時間を越えると、突然、脳の融解(崩壊)が起こり、液化することがあるので、温度と時間の管理を適切に行うことが好ましい。
・透明化追加促進液による処理では、胎児の白色に強調されていた骨化部位の色調も落ちる(透明化する)。従って、骨が見えないほうが良い場合には、透明化追加促進液による処理は有効である。
本発明の透明化生物標本作製用キットは、キット(A)固定液、キット(B)固定液及び透明化促進液、キット(C)固定液及び保存液、または固定液、透明化促進液及び保存液、キット(D)透明化促進液を、それぞれ含むものである。
透明標本保存液の組成の検討
(1)Triton X100およびTween20濃度の検討
標本として、顔料インクを血管内に注入したラット成獣の肝臓を用いた。
2%リン酸緩衝ホルムアルデヒド(パラホルムアルデヒド)、5% Triton X-100、1% KOH混合液にて潅流後、同固定液にて12時間浸漬固定した後、40%エチレングリコール/PB(リン酸塩緩衝液)にて室温保存した。
Triton X-100(Tx100): 20%, 10%, 5%, 1.25%
Tween20(Tw20): 40%, 20%, 10%, 5%, 1.25%
Tx+Tw(各同濃度): 20%, 10%, 5%, 1.25%
Tx-Tw含まないもの: 0%
・赤線よりも左側の処置群で、透明化の効果が確認された。
・Triton X-100, Tween 20単独でも透明化作用は認められるが、両者の混合液では、より低濃度域から透明化作用が認められた。
実施例1
メダカおよびアマガエルの透明化
<条件>
(1)固定
2%リン酸緩衝ホルムアルデヒド(パラホルムアルデヒド)、5% Triton X-100、1% KOH混合液
室温、1時間~オーバーナイト
リン酸緩衝生理食塩水(PBS,pH7.2-7.4)
室温、30分
促進液2:20% Tween20、1% KOH
室温、10分~30分
20% Triton X-100、5% Tween20、20% エチレングリコール(保存液3)
室温、10分~30分
結果を図2に示す。従来の透明化処理法では、透明化完了までに数日から1週間程度必要(固定時間を含まず)。本法では、全身がガラスのように透明になるのが特徴であり、従来のグリセリンやScale試薬による透明標本と比べると、はるかに透明度が高く組織が硬いのが特徴である。図2に示すメダカでは、皮膚、筋肉、内臓はすべて残してある。メラニン色素は残る。図3に示すアマガエルでは、皮膚、筋肉は残してある。内臓が透けて見える。
マウス胎児(胎齢14日)および新生児(生後二日)の透明化
<条件>
(1)固定
2%リン酸緩衝ホルムアルデヒド(パラホルムアルデヒド)、 5% Triton X-100、1% KOH混合液、
室温、1時間~オーバーナイト
リン酸緩衝生理食塩水(PBS,pH7.2-7.4)、
室温、30分
0.3%過酸化水素水、50% アセトン、50% エチレングリコール混合液、
室温、1時間
20% Triton X-100、5% Tween20、20% エチレングリコール(保存液3)
室温、オーバーナイト
10% Triton X-100、10% Tween20、1% KOH(促進液3)
室温、1時間(胎児)、オーバーナイト~数日(新生児)
20% Triton X-100、5% Tween 20、20% エチレングリコール(保存液3)
室温、30分、オーバーナイト~数日(新生児)
結果を図4及び5に示す。従来の透明化処理法では、透明化完了までに数日から1週間程度必要(固定時間を含まず)。本方法は、全身がガラスのように透明になるのが特徴であり、従来のグリセリンやScale試薬による透明標本と比べると、はるかに透明度が高く組織が硬いのが特徴である。透明化と同時に、骨化部位が白く強調される(Tween20またはTween20とエチレングリコール両者の作用と思われる)。Arizarin Red Sを用いた骨染色と同等以上の感度を示す。
マウス成獣消化管組織の透明化(動脈からインク注入)
<条件>
(1)固定
2%リン酸緩衝ホルムアルデヒド(パラホルムアルデヒド)、5% Triton X-100、1% KOH混合液、
室温、1時間~オーバーナイト
リン酸緩衝生理食塩水(PBS,pH7.2-7.4)、
室温、30分
50%アセトン、50%エチレングリコール混合液
室温、数時間~オーバーナイト
20% Triton X-100、5% Tween20、20% エチレングリコール(保存液3)
室温、30分~1時間
結果を図6及び7に示す。
消化管や腹壁など組織の厚さが1-2mm程度の標本の場合、透明標本用固定液にて固定された段階で、組織は十分に透明となる(固定だけで、内部観察が可能)。
小腸壁など、脱脂が不要な部位では、透明化処理全行程3時間以程度で十分である。非常に透明度が高い。
肝臓は、固定時間を長くすることでビリルビンの除去が進み、透明度が上がる。また脱脂の段階で、最終濃度が0.3%となるように過酸化水素水を加えて数時間から一晩漂白することで、組織の着色をほとんど除去することが可能である。
図6に示すマウス成獣の小腸では、 腸絨毛内の毛細血管網が透見される。
図7に示すマウス成獣の肝臓では、肝臓に分布する動脈を細部まで観察することができる。
マウス成獣脳の透明化(その1)
<条件>
(1)固定
2%リン酸緩衝ホルムアルデヒド(パラホルムアルデヒド)、5% Triton X-100、1% KOH混合液、
室温、1時間~オーバーナイト
リン酸緩衝生理食塩水(PBS,pH7.2-7.4)、
室温、30分
0.3% 過酸化水素水、50% アセトン、50% エチレングリコール混合液
室温、24時間
50% アセトン、50% エチレングリコール混合液
室温、数時間~オーバーナイト
20% Triton X-100、5% Tween20、20% エチレングリコール(保存液3)
室温または50℃、24時間~
10% Triton X-100、10% Tween20、1% KOH(促進液3)
室温または50℃、24-48時間
20% Triton X-100、5% Tween20、20% エチレングリコール(保存液3)
結果を図8及び9に示す。
大脳新皮質の灰白質については、漂白、脱脂処理は不要であり、促進液と保存液中で12時間ずつインキュベートすれば、透明化は完成する。目視困難なほど透明化が進む。
ICRマウス成獣(9 wks, ♂)脳の透明化(その2)
<条件>
(1)固定
2%PFA(パラホルムアルデヒド)、5% Triton X100、0.5% KOH、使用まで数か月間固定液中で保存
(2)前処理(洗浄)
20% エチレングリコール、5% Triton X-100、1% KOH、室温、オーバーナイト
(3)保存液(透明化処理1)
20% エチレングリコール、25% Triton X-100、pH 8.5、室温、オーバーナイト(数時間で十分)
(4)保存液(透明化処理4)
20% エチレングリコール、20% Triton X-100、10% DMSO、pH 8.5、室温、オーバーナイト(数時間で十分)
上記(2)、(3)及び(4)の各処理後の結果(写真)を図10に示す。
実施例5では、実施例4におけるグリセロールの使用及び加熱を要しない。(2)の処理においても、透明化は進行するが、(3)の処理後に透明化はほぼ完成する。(4)の処理後は、目視困難なほど透明化が進む。本実施例では、(4)の保存液が10% DMSOを含有するため、標本は膨張するので短時間の観察用に適する。本実施例では脳の例を示したが、肺でも同様に透明化が進むことを確認済みである。肺の場合には、(4)の保存液が10% DMSOを含有しても、標本は膨張しない。
ラット成獣前腕(動脈にインク注入)
標本作製過程は、実施例4に記載の脳の標本作製過程に準じる。結果を図11に示す。
・筋肉をすべて残したまま透明化した。前腕の太い筋肉を通して、骨が明瞭に確認できる。
・筋肉内に分布する血管網が透見できる。
・顕微鏡のフォーカス面における筋線維の走行が確認できる(断層撮影のような観察が可能)。
迅速骨染色法
<条件>
(1)固定:オーバーナイト~数日, 室温
2%リン酸緩衝ホルムアルデヒド(パラホルムアルデヒド), 5% Triton X-100, 1% KOH
・色素はどんどん抜ける。特に赤はよく抜ける。一方、メラニンはなかなか抜けない。
・この段階で十分透明化しておくと、骨染色後の脱色・分別時間が大幅に短縮される。
・皮をはいでおく方が、透明化の時間が短いが、皮を残しておいても十分に透明になる。
・メダカは一晩の固定が適当である。固定液中で、数日でガラス状になる。
・ドジョウは一晩の固定が適当である。しかし体色は十分抜けていない。真っ白になるまで2-3週間、その後に染色する場合、完了まで半日程度かかる。
推奨は、数時間固定してから皮剥ぎすることである。
・固定前に皮剥ぎをしておくと透明化は非常に早いが、皮剥ぎの時にサンプルが壊れやすい。
・数日固定して十分に透明化が進んでいると、皮剥ぎは非常に楽にできる。
・本法では時間をかけると、皮膚も十分に透けるので皮剥ぎは必須ではない。
0.05% アリザンレッドS、1% KOH、1% Tween 20(または1% Triton X-100)
透明化促進液2:20% Tween20、1% KOH
透明化促進液1:20% Triton X-100, 1% KOH, 50℃
透明化促進液3:10% Triton X-100, 10% Tween20, 1% KOH
・哺乳動物は透明化促進液3が良い。
・固定時に標本が半透明になっていれば、染色工程は3-4時間で終了する。
・室温でも洗浄可能であるが、遅く、50℃が好ましい。
・中性~酸性では、急激な組織破壊が起きるため、透明化促進液にはKOHを添加する。
保存液3:20% Triton X-100、5% Tween20、20% エチレングリコール
*保存液3はグリセロールよりも透明度が高い場合があった(特に哺乳動物の組織の場合)
*グリセロール置換による透明化の方法では、メダカ、カエルなどで、固定後~染色終了までの所要時間は数日から1週間である。本法では半日から1日で完了する。
図12に示すメダカでは、半日で完成する。透明度はグリセロール標本よりも高い。
図13に示すエンジェルフィッシュでは、比較的大きい標本でも24時間程度で完成する。
条件1:
固定化: 固定液(2% パラホルムアルデヒド, 5%Triton X-100, 1% KOH混合液)にて固定(室温、オーバーナイト)
保存: 保存液(25% Triton X100, 1% Tween 20, 20% ポリエチレングリコール)にて保存
固定化: 固定液(2% パラホルムアルデヒド)にて固定(室温、オーバーナイト)
透明促進化: 透明促進化液(5%Triton X-100, 1% KOH混合液)(室温、オーバーナイト)
保存: 保存液(25% Triton X100, 1% Tween 20, 20% ポリエチレングリコール) にて保存
固定化: 固定液(4% パラホルムアルデヒド)にて固定(室温、オーバーナイト)
透明促進化: 透明促進化液(5%Triton X-100, 1% KOH混合液)にて固定(室温、オーバーナイト)
保存: 保存液(25% Triton X100, 1% Tween 20, 20% ポリエチレングリコール) にて保存
条件1:
固定化: 固定液(2% パラホルムアルデヒド, 5%Triton X-100, 1% KOH混合液)にて固定(室温、オーバーナイト)
染色: Alizarin red Sにて骨染色
透明促進化: 透明促進化液(20% Tween 20, 1% KOH)、(42℃、オーバーナイト)
透明追加促進化: 透明追加促進化液(グリセロール(100%))
固定化: 固定液(2% パラホルムアルデヒド)にて固定(室温、オーバーナイト)
透明促進化: 透明促進化液(5%Triton X-100, 1% KOH混合液)(室温、オーバーナイト)
染色: Alizarin red Sにて骨染色
透明促進化: 透明促進化液(20% Tween 20, 1% KOH)、(42℃、オーバーナイト)
透明追加促進化: 透明追加促進化液(グリセロール(100%))
固定化: 固定液(4% パラホルムアルデヒド)にて固定(室温、オーバーナイト)
透明促進化: 透明促進化液(5%Triton X-100, 1% KOH混合液)(室温、オーバーナイト)
染色:Alizarin red Sにて骨染色
透明促進化: 透明促進化液(20% Tween 20, 1% KOH)(42℃、オーバーナイト)
透明追加促進化: 透明追加促進化液(グリセロール(100%))
Claims (14)
- 固定液、透明化促進液及び保存液のいずれか1つを含む透明化生物標本作製用キットであって、
前記固定液は、(a)ホルムアルデヒドまたはパラホルムアルデヒド、非イオン系界面活性剤、アルカリ及び緩衝剤を含有する水溶液または(b) ホルムアルデヒドまたはパラホルムアルデヒド、非イオン系界面活性剤、及びアルカリを含有する水溶液であり、
前記透明化促進液は、非イオン系界面活性剤及びアルカリを含有する水溶液であり、
前記保存液は、非イオン系界面活性剤及び多価アルコールを含有する水溶液であって、かつ前記固定液、透明化促進液及び保存液における非イオン系界面活性剤の濃度は、独立に1%以上であるキット。 - 3価アルコール溶液または3価アルコール含有水溶液からなる透明化追加促進液をさらに含む請求項1に記載のキット。
- 生物標本である被透明化物を固定液で処理して、透明化した被透明化物を得る工程(1)を含む透明化生物標本作製方法であって、前記固定液は、(a)ホルムアルデヒドまたはパラホルムアルデヒド、非イオン系界面活性剤、アルカリ及び緩衝剤を含有する水溶液または(b) ホルムアルデヒドまたはパラホルムアルデヒド、非イオン系界面活性剤、及びアルカリを含有する水溶液であり、かつ非イオン系界面活性剤の濃度は、1%以上である方法。
- 生物標本を固定化した被透明化物または請求項3に記載の方法の工程(1)において固定液で処理した被透明化物を透明化促進液で処理して、透明化した被透明化物を得る工程(2)を含む透明化生物標本作製方法であって、前記透明化促進液は、非イオン系界面活性剤及びアルカリを含有する水溶液であり、かつ非イオン系界面活性剤の濃度は、1%以上である方法。
- 前記生物標本を固定化した被透明化物は、生物標本をホルムアルデヒドまたはパラホルムアルデヒドを含み、非イオン系界面活性剤及びアルカリの一方または両方を含有しない水溶液で処理したものである請求項4に記載の方法。
- 請求項3に記載の方法の工程(1)において固定液で処理した被透明化物、または請求項4または5に記載の方法の工程(2)において透明化促進液で処理した被透明化物を保存液で処理して、透明化した被透明化物を得る工程(3) を含む透明化生物標本作製方法であって、前記保存液は、非イオン系界面活性剤及び多価アルコールを含有する水溶液であり、かつ非イオン系界面活性剤の濃度は、1%以上である方法。
- 固定液で処理した被透明化物を漂白、脱脂又は漂白及び脱脂処理した後に、透明化促進液または保存液で処理する請求項4または5に記載の方法。
- 工程(1)~(3)の処理は、独立に、5~60℃の範囲の温度で行う請求項3~7のいずれか1項に記載の方法。
- 工程(1)~(3)のいずれかで得られた透明化した被透明化物を3価アルコール溶液または3価アルコール含有水溶液からなる透明化追加促進液に浸漬し、次いで前記透明化追加促進液に浸漬した後に得られる被透明化物を、請求項5に記載の保存液に保持して、透明化した被透明化物を得る、
請求項3~8のいずれか1項に記載の方法。 - 前記生物標本は、魚類、爬虫類、両生類、鳥類または哺乳類である請求項3~9のいずれか1項に記載の製造方法。
- 前記生物標本は生体組織であり、生体組織は、内臓、脳、または骨である請求項3~10のいずれか1項に記載の製造方法。
- 前記被透明化物は、固定液による処理前または後に染色処理されたものである請求項3~11のいずれか1項に記載の製造方法。
- 前記染色処理が、蛍光染色である請求項12に記載の製造方法。
- 請求項3~13のいずれか1項に記載の方法に用いられる請求項1または2に記載のキット。
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Also Published As
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US20150285718A1 (en) | 2015-10-08 |
EP2926658A1 (en) | 2015-10-07 |
EP2926658A4 (en) | 2016-08-31 |
EP2926658B1 (en) | 2018-08-29 |
JPWO2014069519A1 (ja) | 2016-09-08 |
US11035763B2 (en) | 2021-06-15 |
JP6274443B2 (ja) | 2018-02-07 |
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