• Applied Microscopy
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• 제48권4호
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• pp.87-95
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• 2018

Immunoelectron microscopy using an antigen-antibody reaction in an electron microscope is a very useful tool to identify the components of a tissue in an electron microscope. Many researchers also use immunoelectron microscopy. Nonetheless, immunoelectron microscopy is rarely introduced systematically, and immunoelectron microscopy can be carried out without fully understanding the principles, and cases of poor understanding can often be seen in the vicinity. Therefore, in order to make it easier to understand, we will first introduce the principles of immunoelectron microscopy and describe practical methods.

• 대한수의학회지
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• 제35권3호
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• pp.575-581
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• 1995

Both immunoelectron microscopy(IEM) and immunogold conjugate immunoelectron microscopy (IGC-IEM) techniques were developed for the detection of porcine epidemic diarrhea virus(PEDV) from the feces. Fecal samples were incubated sequentially with anti-PEDV monoclonal antibody(MoAb) and immunogold conjugated goat anti-mouse IgG+IgM. Then negatively stained, mounted on the formvar carbon-coated copper EM grids and observed by the transmission electron microscope. By the direct electron microscopy(DEM), coronavirus particles were observed from 17 cases of total 33 fecal samples of grower pigs and sows. The virons of coronavirus were moderately pleomorphic but mostly spherical, with a diameter ranged from 90 to 190nm. PED virus particles were identified from 15 cases of 17 DEM positive samples by the IEM and IGC-IEM techniques. Aggregates of PED virus coated with specific antibody were seen in fecal samples incubated with homologous anti-PED virus MoAb but not in control samples incubated with anti-TGE virus MoAb. Following incubation with immunogold-conjugated secondary antibody, the gold granules were usually distributed around and among the virus particles and soluble and viral particle-associated antigen. So, IEM and IGC-IEM techniques were proved a rapid and sensitive methods for detection and identification of PED virus from fecal and intestinal contents.

• Applied Microscopy
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• 제28권4호
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• pp.465-475
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• 1998

The cryoprotection, section retrieval and embedding methods were studied for the preservation of ultrastructure of ultracryomicrosections in immunoelectron microscopy. The results obtained were as follows. 1. The cryoprotection of ultrastructure with a mixture containing 1.7 M sucrose and 15% polyvinylpyrrolidone was better than that with 2.3 M sucrose. The stretching caused by surface tension and the electron lucent holes decreased more in the cryosections infused with 2.3 M sucrose than in those with the mixture. 2. The difference between section retrieval solutions in cases of cryoprotection with 2.3 M sucrose was that the destructive .effects such as electron lucent holes and stretching between myofribrils were less in a mixture containing 1% methylcellulose and 2.3 M sucrose than in 2.3 M sucrose. The difference was obscure in the mixture containing 1.7 M sucrose and 15% PVP, but the destructive effects were slightly less in a mixture containing 1% mthylcellulose and 2.3 M sucrose than in 2.3 M sucrose or 1% methylcellulose. 3. The embedding of cryosection on drying with 2% PVA or 2% methylcellulose exhibited some protective effect during observation with transmission electron microscope, but made the ultrastructure more obscure. 4. Mitochondrial membrane and cristae and myofilaments were well delinated in sections infused with 2.3 M sucrose and retrieved with 1% methylcellulose and 2.3 M sucrose. In summary, it is suggested that the cryoprotection with 2.3 M sucrose and section retrieval with a mixture containing 1% methylcellulose and 2.3 M sucrose are good for the ultrastructure of cryosections.

• Applied Microscopy
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• 제23권1호
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• pp.25-34
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• 1993

Prolactin has been reported to be present in the tear film of humans and prolactin-like immunoreactivity has been detected by immunofluorescence in acinar cells of the lacrimal glands of humans and rats. The present study was aimed at clarifying the intracellular distribution of the prolactin-like immunoreactivity, using the electron microscope immunogold technique. The lacrimal gland acinar cells have two types of secretory granules: 1) Secretory granules containing flocculent materials irregularly shaped and are often coalesced. 2) Secretory granules are fairly round and contain homogenous materials of a moderate electron density. The density of the granular content varies even within a single cell. We found prolactin-like reactivity in secretory granules, some smaller cytosolic vesicles, Golgi cisternae and nuclei in acinar cells from intact glands of rat. Our present results are consistent with the conclusion that prolactin is present in lacrimal cells. The presence of prolactin reactivity in the nucleus suggests that prolactin may be a regulatory factor modulating gene expression.

• Applied Microscopy
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• 제24권2호
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• pp.78-92
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• 1994

Lysozyme has been reported to be present in the secretory granules of the Paneth cell, and lysozyme immunoreactivity has been detected by immunogold method in Paneth cells of the intestine of human, mouse and rat. The present study was aimed at clarifying the intracellular distribution and changes of the lysozyme immunoreactivity in rabbit Paneth cell after common bile duct ligation of rabbit, using the electron microscope immunogold technique. Healthy adult rabbits weighing about 2kg body weight were divided into normal and bile duct ligated groups. Common bile duct ligation was performed aseptically under ether anesthesia. Experimental animals were sacrificed on the 1st, the 3rd, the 5th, the 7th and the 14th day after the operation. Mucosal specimens from the intestinal gland of ileum were fixed in 2.5% glutaraldehyde-1.5% paraformaldehyde, followed by 1% osmium tetroxide, embedded in araldite mixture, cut with LKB-V ultratome. Ultrathin sections were placed on parlodion coated nickel grids (200mesh). The section-bearing grids were floated upside down on the added substance in a moist chamber at room temperature except for the primary antibody step, which was at $4^{\circ}C$. Sections were etched with a saturated solution of sodium m-periodate for 60min. After etching, sections were pretreated with 0.02M tris buffered saline (TBS), pH 8.4, with 1% bovine serum albumin (BSA, Sigma) for 60min, then treated polyclonal rabbit anti-human lysozyme (Dakipatts) diluted 1 : 50 in TBS with 0.1% BSA for 20hr. Subsequently, grids were incubated 60min in biotinylated goat anti rabbit IgG (Amersham) diluted 1 : 100 in TBS with 0.1% BSA. After this, sections were incubated 60min on streptavidin gold G10 (Amersham) diluted 1 : 50 in TBS with 0.1% BSA. After each step, the grids were briefly rinsed with TBS with 0.1% BSA. After the strepavidin gold step, the sections were jet washed with distilled water. Counterstain of the sections performed by uranyl acetate and lead citrate, and observed with JEM 100 CX II electron microscope. Observed results were as follow; 1. Secretory granules of mouse Paneth cells have a lysozyme immunoreactivity and also eosinophil leucocyte of rabbit applied for the positive-control stain, are well labeld with gold particles. 2. Normal rabbit Paneth cells have a lysozyme immunoreactivity restricted on the secretory granules. 3. Amount lysosomes containing myelin figures in the Paneth cells were significantly increased from 5th day after the common bile duct ligation. 4. Immunoreactivity of Paneth cell secretory granules were more activated on the 3rd day after the common bile duct ligation as compared with those of the normal animal. But the lysozyme immunoreactivity were decreased from the 5th day after the common bile duct ligation. 5. Considering the above finding, lysozyme contained Paneth cell are affected following of common bile duct ligation, whereas lysosomes containing myelin-figure do not exhibit any immunoreactive relationship with those of secretory granules.