0022-1554/78/2612-i THE JOURNAL

Copyright

121$02.00/0 OF

HISTOCHEMISTRY

© 1978 by The

AND

CYTOCHEMISTRY

Society,

Histochemical

IMMUNOHISTOCHEMICAL RAT MARJORIE Departments

Received

for

LOCALIZATION THYROID

KAREN

WILSON2,

publication

July

OF GLAND’

R. HITCHCOCK3

(M. W., K.R.H.)

of Anatomy

Vol. 26, No. 12, pp. 1121-1124, 1978 Printed in U.S.A.

Inc.

and

THYROID

AND

Pathology

(R.A.D.),

Boston,

Massachusetts

3, 1978,

and

HORMONE

RONALD

Tufts

IN

A. DELELLIS

University

School

of Medicine,

02111

in revised

form

September

5, 1978 (BR

78-121)

and indirect immunofluorescence techniques were used to localize the thyroid triiodothyronine (T3) and thyroxine (Ti) in adult rat thyroid gland. Optimum dilutions of the antisera were established and four tissue fixatives were investigated for usefulness in this technique. Use of antibodies specific for either T3 or T4 resulted in brilliant fluorescence in the colloid pools and apical cytoplasm of follicular cells. In all cases, the adjacent parathyroid gland was devoid of fluorescence. This report demonstrates that these dipeptide hormones can be localized by using immunofluorescence techniques. Direct

hormones

Autoradiographic studies employing ‘25I-labeled antibodies to thyroglobulin (3, 7), immunocytochemical studies with peroxidase-labeled anti-thyroglobulin serum

(7),

as well

as

the

Sternberger

(9)

10%

peroxidase-

anti-peroxidase immunocytochemical technique (8) have been used to localize thyroglobulin in the thyroid gland. All these studies demonstrated that this large storage molecule for the iodinated thyroid hormones is found in the colloid and exists in both large and small

vesicles

in the

thyroid gland. triiodothyronine the thyroglobulin tify

these

of the

follicular

cells

in

the

same

Immunohistochemical

locations techniques

as

a

highly specific tool for identifying the locations of specific antigens in tissue. The objective of this study was to attempt to localize triiodothyronine and thyroxine in adult rat thyroid gland, using immunofluorescence techniques. MATERIALS

Preparation male

of

and

female

River Breeding 200-250 g, were an

intraperitoneal

AND

frozen

METHODS

tissue

sections:

of sodium

pentobarbital.

The thyroid gland, together with the parathyroid, was removed and immediately frozen on dry ice. Tissue sections 6-8 m thick were prepared and stored at -70#{176}Cin a sealed container. Specimens were unfixed or fixed fixatives:

for 10 mm at 4#{176}C in one of the following (a) 95% ethanol, (b) absolute methanol, (c)

Supported

HL15316. 2Present wood Ave., 3Author

by National

Institutes of Health

address: Children’s Hospital, Boston, Massachusetts. to whom reprint requests should

the

changes

of

sections lution

300

slides

were

Grant Long-

be sent.

rinsed

dipped

30 sec

and

2.5%

(d)

(0.1

phosphate-buffered

were for

or

buffer

pH 7.3). All subsequent temperature. To quench

M,

pH

glutaral7.3).

for

10 mm

saline

(PBS,

After

in three 0.01

M,

steps were performed at room possible autofluorescence (4), in a hematoxylin:

rinsed

for

H20

10 min

(1:2)

in three

so-

changes

of PBS. Using

the

technique

of

Coons et al. (2), specimens were incubated with primary antisera for 30 mm in a moist chamber and then rinsed for 15 min in three changes of PBS. Specimens for indirect immunofluorescence were incubated with secondary antiserum for 30 mm and rinsed again for 15 mm in three changes of PBS. Slides were mounted in glycerin: PBS (1:1), coverslipped and examined with a Zeiss fluorescence microscope using UG1 and BG12 excitation filters and Zeiss barrier filters 47 and 65. Photomicrographs were taken using the same exposure times for experimental and matching control specimens.

Adult

CFN derived rats (Charles Laboratories, Wilmington, Mass.), sacrificed with an overdose of ether or Wistar

injection

formalin,

phosphate

Immunofluorescence:

thyro-

provide

buffered

in

fixation,

of the

Since the dipeptide thyroid hormones, and thyroxine, are incorporated into molecule, one might expect to iden-

molecules

globulin.

apex

neutral

dehyde

ity

Antisera: Table 1 summarizes and dilutions of the various

eleven

different

specific studies. binding

antisera,

all

sera

the source, utilized.

raised

in

specificA total of

rabbits

and

for T3 or T4, were screened for use in these Of these, four (Table 1) demonstrated specific patterns in tissue sections. Sera A, B and C were used as primary antisera for indirect immunofluorescence, with serum E as the secondary antiserum. Serum D was used for direct immunofluorescence. All four primary antisera (A-D, Table 1) were raised against T3 or T4 bound to bovine serum albumin (BSA). With antiserum C, some binding in tissue was directed toward BSA producing a diffuse background fluorescence. When this antiserum was absorbed with BSA, this background fluorescence disappeared, leaving the characteristic T3 fluorescent pattern intact (see

1121

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1122

BRIEF

REPORTS

TABLE Source

Anti-T4

B)

187,211 292

Anti-T3

I

Specifications

Lot

Batch

Serum

A)

and

of Sera

1JJ

1489B

1:20-

1489D

1:50

6942A

Utilized

T4’

1:10-

Source

Reactivity

fiIty

T3b

T3, 37% D-T4, 69.6%”

Endocrine

T2,