JP2011203095A - Method of preparing rubber particle sample for electron microscope observation - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method of preparing a rubber particle sample for electron microscope observation.SOLUTION: In the method of preparing the rubber particle sample for electron microscope observation, heavy metal compound is bonded to a rubber particle. In the method of preparing the rubber particle sample, the heavy metal compound is osmium tetroxide. In an observation method of the rubber particle, the rubber particle sample prepared by one of the methods of preparing the rubber particle sample is observed using an electron microscope.

Description

  The present invention relates to a method for preparing a rubber particle sample for observation with an electron microscope.

  Natural rubber is a polymer having elasticity, and is produced by collecting latex called latex, which is mainly produced in cells called a latex tube of rubber tree, and processing it into a desired form ( For example, see Patent Document 1.) The ducts develop several layers a year outside the formation layer in the bark of the rubber tree. The latex is generally collected by scratching (tapping) a rubber tree trunk into a groove shape using a knife or the like and collecting the latex flowing out from the cut milk duct.

  Natural rubber is used widely and in large quantities in various applications as the main raw material for rubber products. For this reason, development of the method of obtaining latex with higher yield is calculated | required. As a method for collecting a larger amount of latex, for example, there is a method in which stimulation of ethylene or ether (2-chloroethylphosphonic acid) is applied to a rubber tree trunk. By applying ethylene or the like to the bark, the latex flowing out from the milk duct is prevented from coagulating at the wound, so that more latex can be collected.

  In the field of medicine and biology, immunoelectron microscopy is known as one of the methods for observing the localization of proteins in a sample such as a biological sample. This method is a combination of immunostaining and observation with an electron microscope. Specifically, first, an antibody that specifically binds to a target protein whose localization is to be examined is used as a primary antibody, and this primary antibody is added to a sample, and the target protein and the primary antibody are bound by an immune reaction. Next, a secondary antibody labeled with gold particles is further bound to the primary antibody bound to the target protein. When the sample prepared in this way is observed with an electron microscope, the localization of the target protein in the sample can be observed as a site labeled with gold particles.

  In natural rubber latex (natural rubber latex), proteins such as REF (rubber elongation factor), which are said to be involved in rubber biosynthesis, are present. By performing immunoelectron microscopy using antibodies against these proteins, the localization of the proteins in the latex can also be observed. For example, in Non-Patent Document 1, the localization of REF on the surface of rubber particles contained in latex is observed by immunoelectron microscopy using a specific antibody against REF. Specifically, first, latex is dropped directly on an observation grid film of a transmission electron microscope (TEM) and dried to place rubber particles in the latex on the observation grid film. An anti-REF antibody is dropped onto the observation grid membrane to bind the REF on the rubber particle surface and the anti-REF antibody by an immune reaction, and then a gold particle-labeled secondary antibody is further bound to the anti-REF antibody. After coating the rubber particles thus prepared with osmium, the rubber particles whose surfaces are labeled with gold particles are observed by observation with a transmission electron microscope under vacuum.

JP 2002-138102 A

Singh, 4 others, Journal of Experimental Botany, 2003, Vol. 54, No. 384, pages 985-992.

  However, since the rubber particles have a low specific gravity and are suspended in the latex, when the rubber particles are placed on a grid film having a diameter of about 3 mm, there is a problem that the sample flows out at the time of dropping and the loss of the sample is large.

  An object of this invention is to provide the method of preparing the rubber particle sample for electron microscope observation.

  As a result of diligent research to solve the above problems, the present inventor previously bound a heavy metal compound to the rubber particles in the latex before dropping the latex onto the observation grid film of the electron microscope, and determined the specific gravity of the rubber particles. It has been found that the rubber particles can be efficiently placed on the observation grid film by increasing the height, and the present invention has been completed.

That is, the present invention
(1) A method for preparing a rubber particle sample for electron microscope observation, characterized in that a heavy metal compound is bonded to the rubber particle,
(2) (a) adding a heavy metal compound to the latex and binding the rubber particles and the heavy metal compound contained in the latex; and (b) centrifuging after the step (a). And a step of recovering rubber particles to which the heavy metal compound is bound from the latex, and a method for preparing a rubber particle sample according to (1),
(3) Further, (c) a step of labeling the rubber particles recovered in the step (b) using an antibody labeled with gold particles, and (d) a centrifugation treatment after the step (c). And a step of removing surplus antibodies from the rubber particles to which the heavy metal compound is bound, and a method for preparing a rubber particle sample according to (2) above,
(4) (a) a step of adding a heavy metal compound to the latex and binding the rubber particles contained in the latex and the heavy metal compound; and (c ′) a rubber in the latex after the step (a). A step of labeling the particles with an antibody labeled with gold particles, and (d ′) after the step (c ′), a centrifugal separation treatment is carried out to remove excess particles from the rubber particles bound with the heavy metal compound. A method for preparing a rubber particle sample according to the above (1), comprising a step of removing the antibody,
(5) The method for preparing a rubber particle sample according to any one of (1) to (4), wherein the heavy metal compound is osmium tetroxide,
(6) A rubber particle observation method characterized by observing a rubber particle sample prepared by the method for preparing a rubber particle sample according to any one of (1) to (5) using an electron microscope,
The purpose is to provide.

  According to the method for preparing a rubber particle sample of the present invention, rubber particles in latex can be efficiently observed with an electron microscope.

In Example 1, it is a photograph figure of the tube after a centrifugation process. In Example 1, it is an optical microscope figure of the black centrifugation residue. In Example 1, it is the figure which showed the measurement result of the particle size distribution of the latex ("osmium addition") which dripped the osmium tetroxide solution, and the latex ("no osmium addition") which did not dripping the osmium tetroxide solution. In Example 1, it is the low vacuum SEM observation image of the collect | recovered rubber particle.

  The method for preparing a rubber particle sample of the present invention is a method for preparing a rubber particle sample for electron microscope observation, characterized in that a heavy metal compound is bound to the rubber particles. By binding the heavy metal compound to the rubber particles, the specific gravity of the rubber particles is increased. For example, when a sample containing rubber particles with an increased specific gravity is dropped on a grid film for a transmission electron microscope, the rubber particles settle, so that the sample can be efficiently placed on the grid film for an electron microscope.

  The heavy metal compound is not particularly limited as long as it is a compound containing a heavy metal capable of increasing the specific gravity of the rubber particles by bonding with the rubber particles. Specific examples of the heavy metal compound include osmium tetroxide, potassium permanganate, uranyl acetate, uranyl formate, phosphotungstic acid, ammonium molybdate, and hafnium chloride. In the present invention, it is preferable to use osmium tetroxide as the heavy metal compound. Osmium tetroxide has a property of adding to a carbon-carbon double bond, and can bind to 1,4-cis-polyisobrene, which is a main component of natural rubber. Specifically, rubber particles and osmium tetroxide can be bound by adding osmium tetroxide to a sample containing rubber particles such as latex.

  In an ordinary electron microscope, an observation sample is coated with osmium tetroxide in order to enhance contrast. For this reason, rubber particles can be observed with high sensitivity by an electron microscope by using osmium tetroxide as the heavy metal compound.

  The rubber particle sample preparation method of the present invention can prepare all rubber particles, but is preferably performed on rubber particles contained in latex (mainly polyisoprene). Such a latex is not particularly limited as long as it is a latex recovered from a latex-producing plant, and may be a latex immediately after being recovered from a latex-producing plant and stored for a certain period after recovery. It may be a thing. In addition, collection | recovery of natural rubber latex from a latex production plant can be performed by a conventional method.

  The latex-producing plant is not particularly limited as long as it is a plant that produces latex (mainly polyisoprene), has a milk duct, and is a plant in which latex is contained in the milk duct. Alternatively, it may be a plant in which latex is contained in the cell space instead of in the duct cells. Examples of such latex-producing plants include, for example, Havea brasiliensis, Meliahot glazovii, Mulberry family, Ficus elasticus, Pana rubber, and Castilla elasticus. Leguminous arabic (Acaccia sengal), Tragant rubber cypress (Astragalus gummifer), Oleander family stag beetle (Dyera costulata), Zanzibar turkey um (Landolphia kirsiel) ca), guayule rubber tree (Parthenium argentatum), rubber dandelion (Taraxacum kok-saghyz), scallops of the categorae family (Palaguium gatata), Mimusops moth Asagao (Cryptostegia grandiflora), Eucommia ulmoides and the like. Among these, para rubber tree, sea rubber tree, rubber dandelion and the like are preferable, and para rubber tree which is widely used as an industrial natural rubber raw material is more preferable.

When a heavy metal compound is bonded to the rubber particles, the rubber particles having an increased specific gravity are likely to settle. Thus, for example, the rubber particles in the latex can be recovered in a state where the rubber particles are concentrated from the latex by performing the following steps (c) and (d).
(A) adding a heavy metal compound to the latex, and bonding the rubber particles contained in the latex and the heavy metal compound;
(B) The process of collect | recovering the rubber particles with which the said heavy metal compound was couple | bonded from the said latex by carrying out the centrifugation process after the said process (a).

  By using the rubber particles collected in the step (b) for electron microscope observation, the rubber particles can be efficiently observed even when the rubber particle concentration is low. Furthermore, after adding water or a suitable buffer to the rubber particles recovered in the step (b), the rubber particles can be washed by recovering the rubber particles by centrifugation or the like.

  In general, in order to observe the protein localization on the rubber particle surface with an electron microscope, it is necessary to label the rubber particle by an immune reaction using an antibody labeled with gold particles (gold particle labeled antibody). . When an antibody was directly added to the latex and an immune reaction was performed, rubber particles having a low specific gravity were difficult to settle, and it was difficult to remove excess antibodies and to wash the rubber particles thereafter. For this reason, latex in a state in which a gold particle-labeled antibody that is bonded to rubber particles and an excess of gold particle-labeled antibody that is not bonded to rubber particles is mixed is placed on the grid film for an electron microscope. There was a problem that it was difficult.

  In contrast, when the rubber particles to which the heavy metal compound is bound are labeled with a gold particle labeled antibody, the excess gold particle labeled antibody can be removed or the rubber particles can be removed after the immune reaction by utilizing the difference in specific gravity. Cleaning can be performed efficiently.

For example, after the step (b), the following steps (c) and (d) are performed, whereby rubber particles from which excess gold particle-labeled antibody has been removed can be recovered.
(C) a step of labeling the rubber particles recovered in the step (b) with a gold particle labeled antibody;
(D) A step of removing surplus antibodies from the rubber particles to which the heavy metal compound is bound by performing a centrifugal separation treatment after the step (c).

Further, an immune reaction for labeling rubber particles with a gold particle-labeled antibody can also be performed in latex. Specifically, after the step (a), the rubber particles from which the excess gold particle labeled antibody has been removed can also be recovered by performing the following steps (c ′) and (d ′).
(C ′) after the step (a), labeling the rubber particles in the latex with an antibody labeled with gold particles;
(D ′) A step of removing excess antibody from the rubber particles to which the heavy metal compound is bound by performing a centrifugal separation treatment after the step (c ′).

  The method of labeling rubber particles using a gold particle labeled antibody is not particularly limited, and any method known in the art may be used. For example, as a gold particle-labeled antibody, a specific antibody against a protein localized on the rubber particle surface is labeled with gold particles, and the gold particle-labeled antibody and rubber particles are bound by an immune reaction, thereby producing gold Rubber particles can be labeled with a particle-labeled antibody. In addition, using a primary antibody as a specific antibody against a protein localized on the rubber particle surface and a gold particle standard antibody in which a secondary antibody that specifically binds to the primary antibody is labeled with gold particles, The rubber particles can be labeled with the gold particle labeled antibody by binding the antibody and rubber particles and then binding the gold particle standard antibody to the primary antibody.

  The rubber particle sample prepared by the rubber particle sample preparation method of the present invention may be observed using either a transmission electron microscope or a scanning electron microscope (SEM). Observation with an electron microscope can be performed by a conventional method. For example, the SEM observation can be performed under a low vacuum (1 to 3000 Pa, more generally 1 to 270 Pa) and an acceleration voltage of 5 to 15 kv.

  In the method for preparing a rubber particle sample of the present invention, rubber particles can be efficiently recovered by a sorting method using a specific gravity difference such as a centrifugal separation process. For this reason, the gold particle labeled antibody and the rubber particle can be directly bonded in the solution, and a suspension of the rubber particle labeled with the gold particle labeled antibody can be obtained. The suspension of rubber particles thus obtained can be dropped on a petri dish or a scanning electron microscope sample stage, and a low vacuum SEM or an atmospheric pressure SEM can be used. In the conventional observation with a transmission electron microscope, the dried rubber particles are observed under vacuum, and thus there is a concern that the shape change such as shrinkage of the rubber particles occurs during the observation. On the other hand, by observing with a scanning electron microscope under low vacuum or atmospheric pressure in a state of containing water, the rubber particles in a state where there is little loss of rubber particles and there is no fear of morphological change are kept for a long time. It becomes possible to observe.

  The rubber particle sample preparation method of the present invention is also applied to a method of collecting the fine particles from a suspension containing fine particles having a light specific gravity other than the rubber particles, and a method of observing the collected fine particles with an electron microscope. Is possible.

  Next, although an experiment example is shown and this invention is demonstrated in detail, this invention is not limited to the following experiment examples.

[Experimental Example 1]
When a 0.2% osmium tetroxide solution was dropped onto a natural rubber latex recovered from para rubber tree and allowed to stand, it was observed that rubber particles were precipitated.
When the latex in which this osmium tetroxide was dropped was centrifuged at 12,000 rpm for 10 minutes, a black centrifugal residue was observed in the system in which the osmium tetroxide solution was dropped. This black centrifugation residue appeared to be rubber particles stained with osmium tetroxide. On the other hand, when a latex treatment was similarly performed on the latex in which osmium tetroxide was not dropped, a white rubber layer was observed in the upper layer. FIG. 1 is a photograph of the tube after centrifugation. The right column of FIG. 1 (“Osmium added”) is the result of the latex to which the osmium tetroxide solution was dropped, and the left column (“No osmium added”) is the result of the latex to which the osmium tetroxide solution was not dropped. Moreover, the upper stage of both rows is before the centrifugation process, and the middle stage shows the figure after the centrifugation process. The lower part is an enlarged view of the tube in the middle part.
When this black centrifugal residue was observed with an optical microscope, it was confirmed that the particles were rubber particles with osmium bound thereto. In FIG. 2, the optical microscope figure of a black centrifugation residue is shown.
Further, the particle size distribution of the black centrifugal residue was measured using a particle size distribution measuring device LS 13320 (manufactured by Beckman Coulter). FIG. 3 is a graph showing the measurement results of the particle size distribution of a latex in which an osmium tetroxide solution was dropped (“osmium added”) and a latex in which an osmium tetroxide solution was not dropped (“no osmium added”). As a result, the particle size distribution was almost the same as that of the rubber particles in the latex in which osmium tetroxide was not dropped. From this measurement result, it was shown that the black centrifugation residue was not what was collect | recovered combining only the specific rubber particle with osmium tetroxide.
From these results, it is clear that rubber particles can be efficiently recovered as a centrifugal separation residue by dropping osmium tetroxide into the latex and binding the rubber particles with osmium tetroxide.

  Further, an anti-REF antibody was dropped onto the collected rubber particles and incubated at room temperature for 1 hour, and then a gold particle-labeled anti-mouse IgG antibody to which gold particles were bound was further added and incubated at room temperature for 1 hour. Then, the centrifugation process was performed and the rubber particle couple | bonded with the antibody was collect | recovered as a centrifugation residue. The recovered rubber particles are dropped onto a scanning electron microscope sample stage in the form of a water-containing suspension and observed with a low-vacuum SEM. The rubber particles bound with the gold particle-labeled anti-mouse IgG antibody are observed. We were able to. FIG. 4 is a low-vacuum SEM observation image of the collected rubber particles. In the figure, some of the small white particles observed on the rubber particle surface are indicated by arrows. These white small particles are gold particle-labeled anti-mouse IgG antibodies bound to the REF protein on the rubber particle surface.

  By using the method for preparing a rubber particle sample of the present invention, rubber particles contained in natural rubber latex and the like can be observed and evaluated efficiently and in detail using an electron microscope. It is particularly useful in the field of production and manufacture.

Claims (6)

A method of preparing a rubber particle sample for electron microscope observation,
A method for preparing a rubber particle sample, characterized in that a heavy metal compound is bonded to rubber particles. (A) adding a heavy metal compound to the latex, and bonding the rubber particles contained in the latex and the heavy metal compound;
(B) After the step (a), a step of recovering rubber particles to which the heavy metal compound is bound from the latex by performing a centrifugal separation process;
The method for preparing a rubber particle sample according to claim 1, comprising: further,
(C) a step of labeling the rubber particles recovered in the step (b) using an antibody labeled with gold particles;
(D) After the step (c), a step of removing the excess antibody from the rubber particles to which the heavy metal compound is bound by centrifuging,
The method for preparing a rubber particle sample according to claim 2, comprising: (A) adding a heavy metal compound to the latex, and bonding the rubber particles contained in the latex and the heavy metal compound;
(C ′) after the step (a), labeling the rubber particles in the latex with an antibody labeled with gold particles;
(D ′) after the step (c ′), a step of removing the excess antibody from the rubber particles to which the heavy metal compound is bound by centrifuging;
The method for preparing a rubber particle sample according to claim 1, comprising:   The method for preparing a rubber particle sample according to any one of claims 1 to 4, wherein the heavy metal compound is osmium tetroxide.   A rubber particle sample prepared by the method for preparing a rubber particle sample according to claim 1 is observed using an electron microscope.