0022-1554/78/2602-0149$02.OO/O THE

JOURNAL

Copyright

OF HISTOCHEMISTRY

© 1978 by The

AND

Histochemical

Vol.

CYTOCHEMLSTRY Society,

Technical

EMBEDDING IMMUNOELECTRON

IN

METHOD OF MICROSCOPIC

SITU

FOR

Y. OHTSUKI,2 Department

of Molecular

Received

and

Carcinogenesis

Hospital

for publication

No.

149-152, 1978 Printed in U.S.A.

2, pp.

Notes

CELLS

L. DMOCHOWSKI,3

M. D. Anderson

26,

Inc.

G. SEMAN

Virology,

The

Tumor

Institute,

and

September

GROWN STUDIES

IN OF

AND

University

J. M. BOWEN of Texas

Houston,

2, 1977, and in revised

BEEM CAPSULES ONCORNAVIRUSES’

System

Texas

form

Cancer

Center,

77030

November

7, 1977

A technique of in situ embedding of cells grown in BEEM capsules has been devised for inimunoelectron microscopic studies of oncornaviruses. As compared to other immunoelectron microscopic procedures, this technique is less time and reagent-consuming. The quality and specificity of this method were tested on well-characterized mouse mammary tumor virus (type B virus) and murine sarcoma virus (type C virus particles). This method gave good results in labeling of the virus particles with ferritin or peroxidase in the cells of mouse tissue cultures. In an application of this method, peroxidase labeling of type B virus particles was obtained in frozen sections of normal prostatic tissues of C3H/Dm and A/Din strain mice treated with rabbit antiserum to mouse mammary tumor virus from A/Dm strain mouse milk. These results indicate that this method is useful and reliable for immunoelectron microscopy studies of oncornaviruses in tissue culture cells and also in frozen sections of tissues. Since

the

initial

description

peroxidase as labels scopic studies utilizing and irnmunoperoxidase applications of these ported in the literature Recently we devised time-saving croscopy.

of

technique The tissue

(5-8, 10-12, a simple,

of the of this

Eppig method

lectron

studies

allows

microscopy entire

BEEM

procedure

capsules.

15, 18). reliable

in

This

the

technique

et

yama pan.

study was supported from the National Cancer of Health, United States address: Department University Medical School, to whom

reprint

requests

prostatic cles,

and

for processing used been

suc-

peroxidase tissue culof normal

in part by Grant Institute, National Public Health Serof Pathology, Okayama, should

containing previously AND

type

B virus

parti-

(2, 3, 14). METHODS

Cells used in the present experiments were: a) type C virus-producing cells of a fibroblastic line (AP-MSV) derived from prostate tissue of an A/Dm strain mouse, and infected with Soehner-Dmochowski strain of murine sarcoma virus (SD-MSV) (1, 17) in vitro, and b) type B virus-producing cells of an epithelial line (C3HMT) derived from spontaneous mammary tumor of a C3H/Dm strain mouse. Antisera tested comprised goat antisera to tweenether disrupted Kirsten (MSV-Ki) murine sarcoma virus and Rauscher leukemia virus (RLV), obtained through the courtesy of Dr. Jack Gruber, Chief, Office of Program Resources and Logistics, Viral Oncology, National Cancer Institute. Other antisera included rabbit antiserum to mouse mammary tumor virus (MMTV) from A/Dm strain mouse milk. Normal rabbit sera were used as controL Ferritin-and peroxidase-conjugated antisera (IgG fraction of goat anti-rabbit IgG and rabbit anti-goat IgG) were purchased from Cappel Laboratories, Downingtown, Pa. and used at 1:4 dilution. The technique, of growing cells in BEEM capsules, used in the present experiments is a slight modification

al. method to immunoe-

has

tissues

as reported

MATERIALS

miused

originally

cessfully tested for the ferritin or labeling of oncornaviruses in mouse ture cells, and also in frozen sections ‘This CA15438 Institutes vice.

and

micro(16) several been re-

for immunoelectron culture procedures

are a modification (4). The application the

ferritin

in immunoelectron both immunoferritin (13) methods, procedures have

Oka700 Jabe sent. 149

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150

TECHNICAL

of the Eppig et al. method (4). Modified points are as follows: Cells are diluted to about 3 to 5 x i0 per ml. After filling the BEEM capsules with cell suspension, capsules are laid in an inverted position in a Petri dish and incubated at 37#{176}C for 1 or 2 days. Growth of cells on the lids was checked under an inverted microscope. Before fixation and processing of the cells, the pointed tips of BEEM capsules are cut away, and the capsules are fastened to a Petri dish with masking tape. Immunoelectron microscopic procedures are as follows: After prefixation in 1% acrolein for 10 mm. at 4#{176}C (10), cells are washed three times in phosphatebuffered saline (PBS) and incubated each for 30 mm. at room temperature with 30-50 il of diluted (1:10 to 1:64) antiserum and with similar amounts of ferritinor peroxidase-conjugated antiserum, and fixed in 3% buffered glutaraldehyde for 30 mm at 4#{176}C. The cells for immunoferritin are then postlixed in 2% buffered osmium tetroxide, dehydrated in graded ethanols and embedded in situ in Epon-Araldite mixture. For immunoperoxidase reaction, the cells are washed briefly in 0.2 M Tris buffer (pH 7.6), incubated in incomplete

NOTES solution of 3-3’diaminobenzidine tetrahydrochloride (2 mg for 10 ml of 0.2 M Tris buffer) for 15 mm, then in complete solution (1 drop of 0.3% hydrogen peroxide added to incomplete solution) for 5 mm in the dark. The cells are postflxed in 1% osmium tetroxide for 30 mm at 4#{176}C, dehydrated and embedded as described above. BEEM capsules are then placed in an oven at 80#{176}C for 1 day, after which the hardened Epon-Araldite blocks are removed from their capsules. After examination

of

the

embedded

cell

layers

under

an

inverted microscope, appropriate fields are selected and the blocks are trimmed by files and razor blades for sectioning with a Sorvall Porter-Blum MT-2B ultramicrotome. Thin sections are stained in uranyl acetate and lead citrate and, after carbon coating, examined in a Siemens Elmiskop 1A electron microscope. Frozen sections of mouse prostate tissues to be tested by the immunoperoxidase method were transferred directly from the blade of the cryostat onto carbon-coated lids of BEEM capsules. The sections were prefixed in periodate-lysine-paraformaldehyde (PLP) solution, containing 2% paraformaldehyde (9,

FIG. MMTV.

1. Ferritin labeling of type B virus particles from C3H-MT cells treated with rabbit Note ferritin labeling of triple-cored particle (arrow). x60,000. FIG. 2. Peroxidase labeling of budding type B virus particle from C3H-MT cells after treatment antiserum to MMTV. x90,000. FIG. 3. Peroxidase labeling of mature type B virus particle treated with rabbit antiserum frozen sections of normal prostate tissue of C3H/Dm mouse. x90,000.

Downloaded from jhc.sagepub.com at Univ of Connecticut / Health Center / Library on May 11, 2015

antiserum with

to rabbit

to MMTV

in

15) and

next

processed

same

the

TECHNICAL

NOTES

culture

immunoelectron

way as tissue

cells.

Type

C virus

AND

particles

type

or

cells both

(Figs. 1 and 2) appear by the immunoferritin methods

mologous goat result

viral

of AP-MSV

particles

of C3H-MT

satisfactorily and by

after

antisera.

treatment

with

Treatment

and

ods. A comparison

with

other

ho-

meth-

methods

reported

or micromicroin BEEM

capsules

have

the

fixatives

(acrolein,

dehyde,

osmium

tetroxide)

hols, propylene capsules. Only and conjugates imens

can

advantages.

All

usual

paraformal-

and

solvents

(alco-

oxide) can be used in BEEM very small amounts of antisera are required, and up to 50 spec-

be

handled

at

one

time.

The

proce-

dure is also very economical since no other containers or vials than the original BEEM capsules used.

capsule onstrated ferritin ferent

Accuracy

and

specificity

procedure compared in Figures 1 and labeling techniques

of this

BEEM

favorably, as dem2, with peroxidase or

of oncornaviruses (15). Results

are

by using difnot only iden-

tical in quality but also can be obtained in a shorter time (15). In an application of this method to an experiment with frozen sections of tissues, immunoperoxidase

labeling

anti-MMTV

of type

serum

C3H/Dm and (Fig. 3) confirmed

in

B virus frozen

A/Dm that

particles sections

of

phologically and immunologically identical the milk-borne MMTV particles. Repeated experiments have confirmed the BEEM capsule procedure works as well PLP

fixative

characterize virus-producing (18)

as in other

techniques viral

or in frozen

This technique to prepare and

immunoelectron

using

the

antigens cells (15), sections may study

(12,

both

the

im-

methods.

same

of

8.

9.

10.

11.

to of cells

15).

be a very convenient way tissue culture cells by

M. Scanlon

Mr.

J.

acknowledged.

CITED

cancer.

Semin

Oncol

3:141,

1976

Dmochowski

In Vitro

micro-

in the cytoplasm virus-infected

of Miss

and

12:65,

1976

5. Hixson DC, Bowen JM: A micromethod for performing ferritin labeling studies on mouse mammary tumor cells. Proc Electron Micros Soc U.S.A. 34:92, 1976 6. Hoshino M, Dmochowski L: Electron microscope study of antigens in cells of mouse mammary tumor cell lines by peroxidase-labeled antibodies in sera of mammary tumor-bearing mice and of patients with breast cancer. Cancer Res 33:2551, 1974 7. Huitric E, Laumomer R, Burtin P, Von Kleist 5, Chavanel G: An optical and ultrastructural study

to

fixative

assistance

L, Ohtsuki Y, Seman G, Maruyama K, Knesek JE, East JL, Bowen JM, Yoshida H, Johnson DE: Search for oncogenic viruses in human prostate cancer. Cancer Treatment Rep 61:119, 1977 4. Eppig JJ, Loiter EH, Waymouth C: Culture of cells in BEEM capsules: A new technique for electron microscopic study of monolayer cultures. 3.

the

that with

Myers,

1. Dmochowski L, East JL, Bowen JM, Lewis ML, Shigematsu T: Studies on tumorigenicity of rat bone tumor virus (SD-MSV-M) in mice, rats and hamsters. Tex Rep Biol Med 30:301, 1972 2. Dmochowski L, Horoszewicz JS; Viral oncology

with

mouse prostate tissues these particles were mor-

technical Brooks

LITERATURE

of prostatic

(6, 8, (7, 12)

on slide glasses, chopped tissues (11), method (5) shows that immunoelectron scopic procedures carried out entirely glutaraldehyde,

Mr.

is gratefully

did not by both

in the literature, by using cell suspensions 15), cultured cells (18) or frozen sections

several

excellent

normal

immunoperoxidase

with

The Chesner,

labeled immu-

the

serum or preimmune rabbit serum in any labeling of these particles

immunoferritin

by

immunoperoxidase

ACKNOWLEDGMENTS

(SD-MSV)

B (MMTV)

noperoxidase

and

DISCUSSION

cells

scopic

microscopy

munoferritin RESULTS

are

151

12.

13.

the

localization

of

carcinoembryonic

antigen

(CEA) in normal and cancerous human rectocoionic mucosa. Lab Invest 34:97, 1976 Kurstak E, Kurstak C: Viral Immunodiagnosis, Chap 1, Academic Press mc, New York, 1974, pp 3-30 McLean 1W, Nakane PK: Periodate-lysine-paraformaldehyde fixative: A new fixative for immunoelectron microscopy. J Histochem Cytochem 22:1077, 1974 Miller MF, Dmochowski L, Maruyama K, Scanlon M, Chesner J: Acrolein prefixation for immunoferritin and immunoperoxidase studies of RNA tumor virus antigens. Proc Electron Micros Sco U.S.A. 32:236, 1974 Muller M, Zotter 5, Kemmer C: Specificity of human antibodies to intracytoplasmic type A particles of the murine mammary tumor virus. J Natl Cancer Inst 56:295, 1976 Nakane PK: Recent progress in the peroxidaselabeled antibody method. Ann N Y Acad Sci 254:203, 1975 Nakane PK, Pierce GB: Enzyme-labeled antibodies: preparation and application for localization of antigens. J Histochem Cytochem 14:929, 1966

Downloaded from jhc.sagepub.com at Univ of Connecticut / Health Center / Library on May 11, 2015

152

TECHNICAL

14. Ohtsuki Y, Seman G, Bowen JM, Dmochowski L: Viruslike particies in human prostate cancer and virus particles in normal prostate of mice. Proc Electron Micros Soc U.S.A. 33:382, 1975 15. Ohtsuki Y, Seman G, Bowen JM, Scanlon M, Dmochowski L: Detection of viral antigens in mouse and rat tumor cells by immunoelectron microscopy following periodate-iysine-paraformaldehyde (PLP) fixation. Proc Electron Micros Soc U.S.A. 34:252, 1976

NOTES 16.

Singer body

SJ: Preparation conjugates. Nature

of an electron-dense 183:1523, 1959

anti-

17. Soehner RL, Dmochowski L: Induction of bone tumors in rats and hamsters with murine sarcoma virus and their cell-free transmission. Nature 224:191, 1969 18. Tabuchi K, Lehman JM, Kirsh WM: Immunocytochemical localization of simian virus 40 T antigen with peroxidase-labeled antibody fragments. J Virol 17:668, 1976

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