MICROSCOPY RESEARCH AND TECHNIQUE 21159-64 (1992)
A Combined Pseudoreplica-lmmunochemical Technique for Research and Diagnostic Virology BLAYNE FRITZ, KENNETH MOORE, AND STANLEY J. NAIDES Department of Internal Medicine and Central Electron Microscopy Research Facility, The University of Iowa College of Medicine, Iowa City, Iowa 52242
KEY WORDS
Vesicular stomatitis virus, Cytomegalovirus, Herpes simplex virus, Human parvovirus B19
ABSTRACT Pseudoreplica and immunochemical techniques were combined in a single protocol for identification of virus in research and clinical specimens. Stock preparations of vesicular stomatitis virus (VSV), cytomegalovirus (CMV), and herpes simplex virus (HSV) were used to develop the technique. Traditional pseudoreplicas of viral stock solutions were prepared but not negatively stained. The virus was then immunolabeled in two stages. Virus-specific polyclonal antisera were used in the first stage; colloidal gold congugated antibodies were used as indicator antibody in the second stage. After immunolabeling, the grids were negatively stained. As a demonstration of the clinical usefulness of this approach, it was employed to antenatally identify human parvovirus B19 particles in ascites from a 22 week gestational fetus with nonimmune hydrops fetalis. The combined pseudoreplica-immunochemical approach offers several advantages over both the pseudoreplica and immunochemical methods when used in isolation. Advantages include relative purification of samples, concentration of virus, morphological preservation, and enhanced diagnostic specificity. INTRODUCTION The pseudoreplica method of staining virus particles for visualization by transmission electron microscopy is a popular techique first developed by Sharp (1960). The advantages of excellent morphological preservation for a broad range of specimens as well as concentration of low titer specimens are distinct advantages over other negative staining techniques (Smith, 1967). Although this protocol alone is employed for many of the research and diagnostic specimens handled by our facility (Smith, 1967; Doane and Anderson, 19871, the combination of pseudoreplica methodology with the highly specific applications of immunochemistry offers greater diagnostic specificity. A protocol combining both pseudoreplica and immunochemistry for identification of vesicular stomatitis virus (VSV),cytomegalovirus (CMV), herpes simplex virus (HSV), and human parvovirus B19, and its clinical applications are described in this report. MATERIALS AND METHODS Pseudoreplica Preparation Sera, ascites, and other thick fluids were diluted in double distilled water (ddHzO)and/or fixative usually at 1:lO to 1:lOO dilution. Feces were likewise diluted in ddH,O and/or fixative. The optimal dilution for a given sample type was determined by comparing serial dilutions in parallel stainings. Watery fluids did not need to be diluted. Stock CMV,, Towne strain, was prepared in human foreskin cell culture. Cell lysates contained 1.1 x 10' pfdml. Stock HSV-1, ATCC strain F, was prepared in human foreskin cell culture. Cell lysates contained 3.3 x lo7 pfdml. Stock VSV, Indiana strain,
0 1992 WILEY-LISS, INC.
was multiple plaque purified and contained 1 x l o 9 pfdml. Virus stocks were undiluted in pseudoreplica preparations. Fifty ml of 2% agarose was prepared and cooled at 4°C in petri dishes. Blocks of 2% agarose were cut from the dishes with a razor and placed on the nonfrosted end of glass microscope slides. A 40 11.1 drop of appropriately diluted sample was placed onto the middle of the agarose block and allowed t o absorb completely. A 40 p1 drop of 0.25%formvar in ethylenedichloride was placed over the dried virus suspension, so that it covered the entire upper surface of the agarose. Excess formvar was removed by tipping the slide and touching a piece of filter paper to the side of the agarose block. All four edges of the formvar-covered block were trimmed with a razor blade so that the formvar was free to separate from the agarose. The slide with the formvar/virus-covered agarose was then lowered into a ddH,O-filled beaker at a 45"angle, making certain the surface tension lifted the formvarhirus film off the agarose to float on the surface of the ddH,O. Nickel grids, 400 mesh, were added dull side down onto the formvar film. Grids were retrieved with a piece of parafilm set onto the grids at an angle so the film stuck to the parafilm and was removed intact. Films were allowed to dry completely on parafilm. Grids were removed with forceps and stored, negative stained or immunostained.
Received May 28, 1991; accepted in revised form October 14, 1991. Address reprint requests to Stanley J. Naides, M.D.,Division of Rheumatology, GH E400, Department of Internal Medicine, The University of Iowa College of Medicine, Iowa City, IA 52242.
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DISCUSSION Immunostaining Pseudoreplica and immunochemical methods each Grids were placed specimen side down on individual drops (25-40 ~ 1 of) goat serum diluted 1:lO in 0.1 M have multiple and specific advantages. The advantages phosphate-buffered saline with 0.1% glycine (PBS/gly- of the combined approach make this technique ideal for cine) for 10 min. The grids were then incubated at room both research and diagnostic applications. The comtemperature on a single drop of appropriately diluted bined approach allows relative purification of dirty first-stage antibody in PBSiglycine for 2 h in a moist- samples and concentration of physiologic titers from, ened chamber. Anti-CMV,, anti-HSV-1, and anti-VSV e.g., serum, fetal ascites, tissue culture, and fecal mapolyclonal antisera were prepared in rabbits and used terial. High morphological preservation and resolution at a dilution of 1:lOO. Anti-B19 monoclonal antibody are achieved. Morphologically simlar viral particles 162-2B, mouse IgM isotype (Biological Products may be differentiated immunologically. Another advantage is that large antigen-antibody Branch, Centers for Disease Control, Atlanta, GA), was used at a dilution of 1:lOO. Grids were washed on 6 complexes cannot form during processing of samples consecutive drops of PBSIglycine, 5 min per drop, then for immunostaining. Viral particles already aggreincubated for 1h on 1drop of second-stage gold labeled gated by endogenous antibody may still be identified. antibody diluted 1:lO with PBS/glycine a t room tem- Viruses which are difficult to culture, e.g., human parperature in moistened chambers. Goat anti-rabbit col- vovirus B19, may be identified; diagnostic results are loidal gold conjugated antibody and goat anti-mouse available within 6 h. The combined approach also alcolloidal gold conjugated antibody (Jannsen Pharma- lows flexibility in handling and storage. Each sample ceutica, Beerse, Belgium) were used at a dilution of prepared can be labeled with numerous antibodies due 1:lO. Grids were washed on 6 consecutive drops of PBS, to multiple grid retrieval. Unlabeled grids can be 5 min per drop. Grids were then washed on 3 consecu- stored and retrieved later for immunolabeling. As a practical approach, one or a few grids from each preptive drops of water, 2 min per drop, then wick dried. aration can be negatively stained and examined before immunolabeling, eliminating wasted time and reNegative Staining agents should viral particles not be present. Grids were placed on one drop of 2.0% aqueous phosphotungstic acid for 2 min or 5% aqueous uranyl aceACKNOWLEDGMENTS tate for 1min. Excess stain was pulled off with a corner The authors thank Dr. Jose Rodriguez for the generof filter paper. The grid and specimen were placed spec- ous gift of CMV , HSV-1, and VSV viral stocks, and imen side up to air dry. Grids were then examined for rabbit polyclonaf antisera, and Ms. Kathy Eckrich for negatively stained, formvar-embedded viral particles secretarial assistance in preparation of the manuscript. with colloidal gold label. RESULTS VSV, CMV or HSV were labeled in a two-step procedure using virus-specific rabbit antiserum followed by goat anti-rabbit antibody conjugated to 10 nm colloidal gold (VSV, Fig. 1A-C; CMV, Fig. 2A-C; HSV, Fig. 3A-C). Virus particles were visualized by negative staining after colloidal gold conjugated antibody labeling. Immunolabeling was specific in that colloidal gold labeling did not occur when irrelevant antiserum was used a s the first-stage antibody (VSV, Fig. 1D,E; CMV, Fig. 2D,E; HSV, Fig. 3D,E). As a n additional control for nonspecific labeling by the second-stage colloidal gold conjugated goat anti-rabbit antibody, pseudoreplicas of virus were prepared and immunogold labeled in the absence of first-stage antigody. Such preparations lacking a first-stage antibody did not label (VSV, Fig. 1F; CMV, Fig. 2F; HSV, Fig. 3F). Fetal ascites from a 22 week gestational fetus with nonimmune hydrops fetalis was obtained antenatally for diagnosis. Pseudoreplica preparations demonstrated approximately 23 nm nonenveloped viral particles morphologically consistent with parvovirus B19. Immunogold labeling of ascites with 5 nm colloidal gold conjugated antibody established the diagnosis of parvovirus B19 infection as previously reported by Naides and Weiner (1989) (Fig. 4A). Preparations with a n irrelevant first-stage antibody (Fig. 4B), or without a first-stage antibody (Fig. 4C), did not label.
~
Fig. 1. (See p. 61.) A X : VSV, pseudoreplica processed and primary stage labeled with rabbit anti-VSV polyclonal antiserum. A-C were second-stage labeled with 10 nm colloidal gold bound to goat anti-rabbit antibody at dilution. D: VSV, pseudoreplica processed and primary-stage labeled with rabbit anti-CMV polyclonal antiserum. E: VSV, pseudoreplica processed and primary-stage labeled with rabbit anti-HSV polyclonal antiserum. F: VSV, pseudoreplica processed and no primary-stage antibody. D-F were second-stage labeled with 10 nm colloidal gold bound to goat anti-rabbit antibody. Negative staining with phosphotungstic acid. Original magnification x 50,000; pictured here x 95,000. Fig. 2. (See p. 62.) A-C: CMV pseudoreplica processed and primary-stage labeled with rabbit anti-CMV polyclonal antiserum; second-stage labeled with 10 nm colloidal gold bound to goat anti-rabbit antibody. D: CMV, pseudoreplica processed and primary-stage labeled with rabbit anti-VSV polycional antiserum. E CMV, pseudoreplica processed and primary-staged labeled with rabbit anti-HSV polyclonal antiserum. F CMV, pseudoreplica processed and no primarystage antibody. D-F were second-stage labeled with 10 nm colloidal gold bound to goat anti-rabbit antibodies. Negative staining with phosphotungstic acid. Original magnification x 30,000; pictured here X 54,000. Fig. 3. (See p. 63.) A X : HSV, pseudoreplica processed and primary-stage labeled with rabbit anti-HSV polyclonal antiserum; second-stage labeled with 10 nm colloidal gold bound to goat anti-rabbit antibody. D: HSV, pseudoreplica processed and primary-stage labeled with rabbit anti-VSV polyclonal antiserum. E: HSV, pseudoreplica processed and primary-stage labeled with rabbit anti-CMV polyclonal antiserum. F: HSV, pseudoreplica processed and no primary-stage antibody. D-F were second-stage labeled with 10 nm colloidal gold bound to goat anti-rabbit antibody. Negative staining with phosphotungstic acid. Original magnification X 50,000; pictured here X 87,500.
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Fig. 2.
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Fig. 4. A: Pseudoreplica processed fetal ascites (diluted 1:lOO in PBS) labeled with primary-stage anti-B19 monoclonal antibody 1622B (mouse IgM) and second-stage 5 nm colloidal gold conjugated goat anti-mouse immunoglobulin antibody. Original magnification x 100,000; pictured here x 190,000,B: Fetal ascites pseudoreplica processed and labeled with anti-CMV antiserum as first-stage anti-
This work was supported in part by the March of Dimes Birth Defects Foundation, the ~~~~i~~~philosophical the Department Of Society, the Arthritis Veterans Affairs Research Fund, and The University of Iowa College of Medicine and the Graduate College.
REFERENCES Doane F.W., and Anderson N. (1987). Electron Microscopy in Diagnostic Virology. A Practical Guide and Atlas. Cambridge University Press, Cambridge, pp. 18-19.
body, followed by 5 nm colloidal gold conjugated goat anti-rabbit immunoglobulin antibody. Original magnification x 50,000; pictured here X 95,000. C: Pseudoreplica processed fetal ascites with no primary antibody followed by 5 nm colloidal gold conjugated goat antimouse immunoglobulin antibody. Negative staining with uranyl acetate. Original magnification x 50,000; pictured here x 126,000.
Naides, S.J., and Weiner, C.P. (1989). Antenatal diagnosis and palliative treatment of non-immune hydrops fetalis secondary to fetal parvovirus B19 infection. Prenatal Diagnosis, 9:105-114. Sharp, D. (1960). Sedimentation counting of particles via electron microscopy. In: Proceedings of the IVth International CongTess of Electron Microscopy, Vol. 11, Springer, Berlin, pp. 542-548. Smith K.O. (1967). Identification of viruses by electron microscopy. In: Methods in Cancer Research. H. Busch, ed. Academic Press, New York, pp. 545-572.
A Combined Pseudoreplica-lmmunochemical Technique for Research and Diagnostic Virology BLAYNE FRITZ, KENNETH MOORE, AND STANLEY J. NAIDES Department of Internal Medicine and Central Electron Microscopy Research Facility, The University of Iowa College of Medicine, Iowa City, Iowa 52242
KEY WORDS
Vesicular stomatitis virus, Cytomegalovirus, Herpes simplex virus, Human parvovirus B19
ABSTRACT Pseudoreplica and immunochemical techniques were combined in a single protocol for identification of virus in research and clinical specimens. Stock preparations of vesicular stomatitis virus (VSV), cytomegalovirus (CMV), and herpes simplex virus (HSV) were used to develop the technique. Traditional pseudoreplicas of viral stock solutions were prepared but not negatively stained. The virus was then immunolabeled in two stages. Virus-specific polyclonal antisera were used in the first stage; colloidal gold congugated antibodies were used as indicator antibody in the second stage. After immunolabeling, the grids were negatively stained. As a demonstration of the clinical usefulness of this approach, it was employed to antenatally identify human parvovirus B19 particles in ascites from a 22 week gestational fetus with nonimmune hydrops fetalis. The combined pseudoreplica-immunochemical approach offers several advantages over both the pseudoreplica and immunochemical methods when used in isolation. Advantages include relative purification of samples, concentration of virus, morphological preservation, and enhanced diagnostic specificity. INTRODUCTION The pseudoreplica method of staining virus particles for visualization by transmission electron microscopy is a popular techique first developed by Sharp (1960). The advantages of excellent morphological preservation for a broad range of specimens as well as concentration of low titer specimens are distinct advantages over other negative staining techniques (Smith, 1967). Although this protocol alone is employed for many of the research and diagnostic specimens handled by our facility (Smith, 1967; Doane and Anderson, 19871, the combination of pseudoreplica methodology with the highly specific applications of immunochemistry offers greater diagnostic specificity. A protocol combining both pseudoreplica and immunochemistry for identification of vesicular stomatitis virus (VSV),cytomegalovirus (CMV), herpes simplex virus (HSV), and human parvovirus B19, and its clinical applications are described in this report. MATERIALS AND METHODS Pseudoreplica Preparation Sera, ascites, and other thick fluids were diluted in double distilled water (ddHzO)and/or fixative usually at 1:lO to 1:lOO dilution. Feces were likewise diluted in ddH,O and/or fixative. The optimal dilution for a given sample type was determined by comparing serial dilutions in parallel stainings. Watery fluids did not need to be diluted. Stock CMV,, Towne strain, was prepared in human foreskin cell culture. Cell lysates contained 1.1 x 10' pfdml. Stock HSV-1, ATCC strain F, was prepared in human foreskin cell culture. Cell lysates contained 3.3 x lo7 pfdml. Stock VSV, Indiana strain,
0 1992 WILEY-LISS, INC.
was multiple plaque purified and contained 1 x l o 9 pfdml. Virus stocks were undiluted in pseudoreplica preparations. Fifty ml of 2% agarose was prepared and cooled at 4°C in petri dishes. Blocks of 2% agarose were cut from the dishes with a razor and placed on the nonfrosted end of glass microscope slides. A 40 11.1 drop of appropriately diluted sample was placed onto the middle of the agarose block and allowed t o absorb completely. A 40 p1 drop of 0.25%formvar in ethylenedichloride was placed over the dried virus suspension, so that it covered the entire upper surface of the agarose. Excess formvar was removed by tipping the slide and touching a piece of filter paper to the side of the agarose block. All four edges of the formvar-covered block were trimmed with a razor blade so that the formvar was free to separate from the agarose. The slide with the formvar/virus-covered agarose was then lowered into a ddH,O-filled beaker at a 45"angle, making certain the surface tension lifted the formvarhirus film off the agarose to float on the surface of the ddH,O. Nickel grids, 400 mesh, were added dull side down onto the formvar film. Grids were retrieved with a piece of parafilm set onto the grids at an angle so the film stuck to the parafilm and was removed intact. Films were allowed to dry completely on parafilm. Grids were removed with forceps and stored, negative stained or immunostained.
Received May 28, 1991; accepted in revised form October 14, 1991. Address reprint requests to Stanley J. Naides, M.D.,Division of Rheumatology, GH E400, Department of Internal Medicine, The University of Iowa College of Medicine, Iowa City, IA 52242.
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DISCUSSION Immunostaining Pseudoreplica and immunochemical methods each Grids were placed specimen side down on individual drops (25-40 ~ 1 of) goat serum diluted 1:lO in 0.1 M have multiple and specific advantages. The advantages phosphate-buffered saline with 0.1% glycine (PBS/gly- of the combined approach make this technique ideal for cine) for 10 min. The grids were then incubated at room both research and diagnostic applications. The comtemperature on a single drop of appropriately diluted bined approach allows relative purification of dirty first-stage antibody in PBSiglycine for 2 h in a moist- samples and concentration of physiologic titers from, ened chamber. Anti-CMV,, anti-HSV-1, and anti-VSV e.g., serum, fetal ascites, tissue culture, and fecal mapolyclonal antisera were prepared in rabbits and used terial. High morphological preservation and resolution at a dilution of 1:lOO. Anti-B19 monoclonal antibody are achieved. Morphologically simlar viral particles 162-2B, mouse IgM isotype (Biological Products may be differentiated immunologically. Another advantage is that large antigen-antibody Branch, Centers for Disease Control, Atlanta, GA), was used at a dilution of 1:lOO. Grids were washed on 6 complexes cannot form during processing of samples consecutive drops of PBSIglycine, 5 min per drop, then for immunostaining. Viral particles already aggreincubated for 1h on 1drop of second-stage gold labeled gated by endogenous antibody may still be identified. antibody diluted 1:lO with PBS/glycine a t room tem- Viruses which are difficult to culture, e.g., human parperature in moistened chambers. Goat anti-rabbit col- vovirus B19, may be identified; diagnostic results are loidal gold conjugated antibody and goat anti-mouse available within 6 h. The combined approach also alcolloidal gold conjugated antibody (Jannsen Pharma- lows flexibility in handling and storage. Each sample ceutica, Beerse, Belgium) were used at a dilution of prepared can be labeled with numerous antibodies due 1:lO. Grids were washed on 6 consecutive drops of PBS, to multiple grid retrieval. Unlabeled grids can be 5 min per drop. Grids were then washed on 3 consecu- stored and retrieved later for immunolabeling. As a practical approach, one or a few grids from each preptive drops of water, 2 min per drop, then wick dried. aration can be negatively stained and examined before immunolabeling, eliminating wasted time and reNegative Staining agents should viral particles not be present. Grids were placed on one drop of 2.0% aqueous phosphotungstic acid for 2 min or 5% aqueous uranyl aceACKNOWLEDGMENTS tate for 1min. Excess stain was pulled off with a corner The authors thank Dr. Jose Rodriguez for the generof filter paper. The grid and specimen were placed spec- ous gift of CMV , HSV-1, and VSV viral stocks, and imen side up to air dry. Grids were then examined for rabbit polyclonaf antisera, and Ms. Kathy Eckrich for negatively stained, formvar-embedded viral particles secretarial assistance in preparation of the manuscript. with colloidal gold label. RESULTS VSV, CMV or HSV were labeled in a two-step procedure using virus-specific rabbit antiserum followed by goat anti-rabbit antibody conjugated to 10 nm colloidal gold (VSV, Fig. 1A-C; CMV, Fig. 2A-C; HSV, Fig. 3A-C). Virus particles were visualized by negative staining after colloidal gold conjugated antibody labeling. Immunolabeling was specific in that colloidal gold labeling did not occur when irrelevant antiserum was used a s the first-stage antibody (VSV, Fig. 1D,E; CMV, Fig. 2D,E; HSV, Fig. 3D,E). As a n additional control for nonspecific labeling by the second-stage colloidal gold conjugated goat anti-rabbit antibody, pseudoreplicas of virus were prepared and immunogold labeled in the absence of first-stage antigody. Such preparations lacking a first-stage antibody did not label (VSV, Fig. 1F; CMV, Fig. 2F; HSV, Fig. 3F). Fetal ascites from a 22 week gestational fetus with nonimmune hydrops fetalis was obtained antenatally for diagnosis. Pseudoreplica preparations demonstrated approximately 23 nm nonenveloped viral particles morphologically consistent with parvovirus B19. Immunogold labeling of ascites with 5 nm colloidal gold conjugated antibody established the diagnosis of parvovirus B19 infection as previously reported by Naides and Weiner (1989) (Fig. 4A). Preparations with a n irrelevant first-stage antibody (Fig. 4B), or without a first-stage antibody (Fig. 4C), did not label.
~
Fig. 1. (See p. 61.) A X : VSV, pseudoreplica processed and primary stage labeled with rabbit anti-VSV polyclonal antiserum. A-C were second-stage labeled with 10 nm colloidal gold bound to goat anti-rabbit antibody at dilution. D: VSV, pseudoreplica processed and primary-stage labeled with rabbit anti-CMV polyclonal antiserum. E: VSV, pseudoreplica processed and primary-stage labeled with rabbit anti-HSV polyclonal antiserum. F: VSV, pseudoreplica processed and no primary-stage antibody. D-F were second-stage labeled with 10 nm colloidal gold bound to goat anti-rabbit antibody. Negative staining with phosphotungstic acid. Original magnification x 50,000; pictured here x 95,000. Fig. 2. (See p. 62.) A-C: CMV pseudoreplica processed and primary-stage labeled with rabbit anti-CMV polyclonal antiserum; second-stage labeled with 10 nm colloidal gold bound to goat anti-rabbit antibody. D: CMV, pseudoreplica processed and primary-stage labeled with rabbit anti-VSV polycional antiserum. E CMV, pseudoreplica processed and primary-staged labeled with rabbit anti-HSV polyclonal antiserum. F CMV, pseudoreplica processed and no primarystage antibody. D-F were second-stage labeled with 10 nm colloidal gold bound to goat anti-rabbit antibodies. Negative staining with phosphotungstic acid. Original magnification x 30,000; pictured here X 54,000. Fig. 3. (See p. 63.) A X : HSV, pseudoreplica processed and primary-stage labeled with rabbit anti-HSV polyclonal antiserum; second-stage labeled with 10 nm colloidal gold bound to goat anti-rabbit antibody. D: HSV, pseudoreplica processed and primary-stage labeled with rabbit anti-VSV polyclonal antiserum. E: HSV, pseudoreplica processed and primary-stage labeled with rabbit anti-CMV polyclonal antiserum. F: HSV, pseudoreplica processed and no primary-stage antibody. D-F were second-stage labeled with 10 nm colloidal gold bound to goat anti-rabbit antibody. Negative staining with phosphotungstic acid. Original magnification X 50,000; pictured here X 87,500.
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Fig. 2.
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Fig. 4. A: Pseudoreplica processed fetal ascites (diluted 1:lOO in PBS) labeled with primary-stage anti-B19 monoclonal antibody 1622B (mouse IgM) and second-stage 5 nm colloidal gold conjugated goat anti-mouse immunoglobulin antibody. Original magnification x 100,000; pictured here x 190,000,B: Fetal ascites pseudoreplica processed and labeled with anti-CMV antiserum as first-stage anti-
This work was supported in part by the March of Dimes Birth Defects Foundation, the ~~~~i~~~philosophical the Department Of Society, the Arthritis Veterans Affairs Research Fund, and The University of Iowa College of Medicine and the Graduate College.
REFERENCES Doane F.W., and Anderson N. (1987). Electron Microscopy in Diagnostic Virology. A Practical Guide and Atlas. Cambridge University Press, Cambridge, pp. 18-19.
body, followed by 5 nm colloidal gold conjugated goat anti-rabbit immunoglobulin antibody. Original magnification x 50,000; pictured here X 95,000. C: Pseudoreplica processed fetal ascites with no primary antibody followed by 5 nm colloidal gold conjugated goat antimouse immunoglobulin antibody. Negative staining with uranyl acetate. Original magnification x 50,000; pictured here x 126,000.
Naides, S.J., and Weiner, C.P. (1989). Antenatal diagnosis and palliative treatment of non-immune hydrops fetalis secondary to fetal parvovirus B19 infection. Prenatal Diagnosis, 9:105-114. Sharp, D. (1960). Sedimentation counting of particles via electron microscopy. In: Proceedings of the IVth International CongTess of Electron Microscopy, Vol. 11, Springer, Berlin, pp. 542-548. Smith K.O. (1967). Identification of viruses by electron microscopy. In: Methods in Cancer Research. H. Busch, ed. Academic Press, New York, pp. 545-572.
