THE

JOURNAL

Copyright

H ISTOCHEMISTRY

OF

© 1976

by The

C YTOCHEMISTR

AND

Histochemical

ULTRASTRUCTURAL THE

Departments

Northwestern

of Internal

Center,

42nd

and

Dewey

Avenue,

Hospital, Received

for

and

4101

School,

Biochemistry Omaha,

publication

(R.

Nebraska

15,

Chicago,

B.

T.),

1976

and

Illinois

University

68105

and

Omaha,

Avenue,

March

CHARACTERIZATION PITUITARIES’

HUMAN

Medical

Woolworth

1976

in U.S.A.

OF

AND R. B. TOBIN

University

Medicine

1131-1139. Printed

IMMUNOCYTOCHEMICAL THYROTROPH IN RAT AND

of Anatomy,

11. PP

Inc.

G. C. MORIARTY2 Department

24, No

Vol.

Society.

Veterans

Nebraska

in revised

60611

(G.

of Nebraska

C. M.)

and

Medical

Administration

68105

form

July

19,

1976

The 11 chain of thyroid stimulating hormone (TSH) was localized in normal rat and human pituitaries with the unlabeled antibody-peroxidase-antiperoxidase complex technique and antisera to rat or bovine TSHII. In Araldite embedded tissues, immunoreactivity of TSH was best preserved after fixation with 1% glutaraldehyde or picric acid formaldehyde. In the TSH cells, the immunocytochemical stain was located on granules. However, there was a variation in staining of individual granules, and among the population of granules. Rat TSH cells were ovoid or angular to stellate, and contained granules ranging in size from 60-175 nm. The human TSH cell was polyhedral, and contained scattered grantiles of 150-300 nm diameter. In both species, granule distribution was either in 1-2 rows at the periphery, or scattered throughout the cytoplasm with some concentration in cytoplasmic processes. TSH cells in female rats in estrous contained more granules than those of other stages. TSH cells were distinguished from adrenocorticotropic hormone cells and luteinizing hormone cells on the basis of granule size and distribution, and cell shape.

The

of

thyroid

the

ovoid

stimulating

hormone

rat

have

been

cells

that

contain

scattered

at

Costoff,

its

the diameter

or

granules,

(4-6,

In

cells

angular

cytoplasm

periphery

granule

as

small

throughout

trated

(TSH)

described

According

ranges

from

to

as

studies,

were

classical sera

the anti

We

have

TSH

cells

hormone

from (LH)

cells

and that

the

cells

However,

reported

cells

contain

the

adrenocorticotropic

as

granules

morphology

their

irregular

by

Grants

from

the

Bly

prelude

of which

l3

chain

of

and characterize This investigato

in

the

studies

of

TSH

thyroidectomy will

be

cells

reported

in a

paper.

from

tissues

be mistaken

(ACTH)

hormone

a

results

dehydration

TSH

granules, may

to identify and humans.

packaging

tech-

to

AND

METHODS

Polysciences

or

EM

Sciences,

picric

acid formaldehyde (10). or 1-4% p-formaldehyde, for 1 hr at room temperature. Glutaraldehyde and pformaldehyde fixatives were made with 0.1 M Sorenson’s phosphate buffer, pH 7.4. Following ethanol

luteinizing

of

of

anti-

might

cells

and were

in glycol

propylene

embedded

methacrylate

oxide either

in

infiltration, Araldite

the 6005

or

(17).

Ultrathin sections were cut and mounted on nickel grids. They were then etched in 10% H202, washed in water and stained on drops of the following solutions: Step 1: Normal goat serum, 1:100 dilution, 3 mm. Step 2: Anti-TSH(i serum, (1:10,000 anti-bovine TSHI or 1:25,000 anti-rat TSH), 48 hr 4#{176}C. Phosphate buffer wash. Step 3: Repeat step 1. Step 4: Goat anti-rabbit immunoglobulin G (IgG),

(9). ‘Funded

the

antisera

Pituitaries from male and female rats were minced in 1 mm3 pieces, and immersed in 1 or 2% glutaraldehyde (made from vials of 70% glutaraldehyde),

distinguishing

basis

arranged few

as

and

obtained

in

because

peripherally

served

companion

by

used

which

identified on

tion

immunocytochemical

MATERIALS

described

they

difficulty

cells

used in rats

immunocytochemical

to

entire TSH molecule, a chain antibodies.

Furthermore,

shape for

by

similar

cytologists. to

contain

(9).

identified

TSH

were cells

(14),

mgi, with a mean of 80 mg. Nakane (15) and Kawari and Nakane (7) reported that thyrotrophs,

specific

storage

40-150

study,

and

TSH

either

or concen-

8).

this

niques

Memorial

Research Fund, Omaha Veterans Administration Hospital, and NIH HD08842. 2 Send reprint requests to: Gwen C. Moriarty, Department of Anatomy, Northwestern University Medical School, Chicago, Ill. 60611. to the entire TSH molecule may contain a chain antibodies which would react with gonadotropin a chains (9).

1:100

Step

dilution, 5: Repeat

3 mm. step

1131

Downloaded from jhc.sagepub.com by guest on March 13, 2015

Phosphate 1.

buffer

wash.

1132

MORIARTY

Step

6:

dilution,

Peroxidase-ant

3 mm.

iperoxidase

(Obtained

from

solution.

Dr.

AND

1:100

L. A. Sternberger,

or Cappell Laboratories, Downington, Pa.). Phosphate buffer wash. Step 7: Drops of phosphate buffer until diaminobenzidine solution is made. The diaminobenzidine solution is made as in previous reports (9, 11, 12), and is kept moving during the reaction process of 3-4 mm. Water wash. Step 8: Four percent Os04, 8-10 mm. Water wash. Water is distilled-deionized. All solutions, including antisera, are millipore filtered. This technique is detailed in previous publications (18,

9-12).

TSHI3

chain

antisera

were

employed.

One

was a gift from Dr. J. G. Pierce, and was made to bovine TSHfl (bTSHfl). The other two were obtained from The National Institute of Arthritis, Metabolism Digestive

from

Dr.

Diseases

A.

F.

Control

(NIAMDD),

Parlow;

TSH1 (rTSHfl) (hTSHfl).

and

one

the

and

was

other

were

produced

with

gifts

with

human

rat

TSHI3

procedures

determined

were

performed

and

quantitat-

as

in a previous

Specificity

report

thyrotrophs

experiments

TSH whereas mone

The

and

No

to

provided

Absorption

stock of

(NIAMDD 1 g (FSH)

of had

anti-rTSHfl

this

was

I-i) LH no

or

more

of

staining,

follicle-stimulating

significant

was

10 ng

effect

served

specific

hor1).

than

the

posthe

anti-

by

in

of

glutaraldehyde

that

few tissue

than

For

the

of

1%],

both

immunoreactivity,

hyde were

or paraformaldehyde picric satisfactory. Extragranular

as

those

found

in

and

and

plasmic

of

cells,

the

the

that

(Fig.

arranged

in

TSH

cell

or

in

1%

male

ovoid

to

small

cyto-

in diameter In

of

the

at

they

more

from

some

distributed

However,

two

by

were

nm).

were

3).

endoplasmic only

ranged 130

granules

riphery

glutaralde-

scattered

(average

(Table

acid (PAF, 10) antigens, such

cells

contained

nm

1%

rough

TSH

granules

50-175

2%

preserved

Characterization female rats:

angular,

of

sacs

were

concentra-

ultrastructure

either

reticulum (14), glutaraldehyde.

or

than

and

was

concentrations

higher

preservation

the

is pre-

Staining

with

higher

of p-formaldehyde

(10),

molecule

fixatives.

fixed

serum if goat

stain. on TSH

of LH

TSH

the

relatively

observed

rabbit or

omitted from the and embedding of

not

were

rows,

and

the

peoften

unevenly

distributed.

(Figs.

diameter and

cells.

of the the

of

these

in

cells

classical

However,

when

the

size

measured,

they

fit

the

For

example,

the

in Figures size

contained

250 nm

Some

more nuthe cyto-

described

granules

maximum

generally

same

was

TSH

for

5). cell

(9, 10).

granules

criteria

granules were throughout

and LH

studies

the

of

4 the

was

average

was

160

granules

that

nm.

130 LH

nm, cells

200-

were

in diameter. stained

group

previous

(Fig.

intensity, in

Unlike

immunoreactivity

We

for further

abolished

staining

seen if normal the anti-TSHf,

for

IgG was

cytologic

with

employed

contamination

Effect of fixation immunoreactivity:

characteris-

solutions antisera

in

TSH

staining substituted

was

resembled

gonadotrophs

routinely

gens.

anti-bTSHf3

(10). After absorption of a 1:1000 dilution with 130 zg human chorionic gonadotrophin/mi to remove a chain antibodies that would cross-react with gonadotropins (9), and further dilution to 1:10,000 only TSH cells were stained. These tests.

due

In other TSH cells, merous, and scattered

(10).

The

2). It was

reduction

sibly

plasm

of the reaction: with the morphologic

cells of

significant

I).

RESULTS

tics

(Fig.

at a 1:10,000 or 1:25,000 dilution, and it stained only TSH cells. Staining was significantly reduced when the antiserum was absorbed with 10 pg of TSH (Fig. 2), and abolished with 100 pg of TSH. Ten thousand times more FSH and LH failed to abolish staining; however, there was a

tions

ed as in a previous investigation (10, 13). Several types of controls were utilized. First, a 1:1000 dilution of normal rabbit serum was substituted for the primary anti-serum (anti-TSHfl). Second, the second antibody (goat anti-rabbit IgG) was omitted from the sequence. Third, the anti-TSH$ was absorbed with either LH, TSH or FSH for 2 days prior to its use in the stain. This last test was run three times. In all of our controls, the staining intensity was analyzed densitometrically as previously described (13). In each test, 50 to 100 secretory granules were analyzed per a given absorption. Thus, each point on our immunoabsorption curves (Figs. 1 and 2) represents the normalized staining intensity of at least 100 granules. The animals studied included four male and 12 female rats. The stage of the estrous cycle was

stained

anti-bTSHf3

anti-rabbit

Three

and

TOBIN

differences

sections of cycling

report

with

of female

(10),

and

each

stage

that TSH cells in estrous tained more granules (Fig.

Downloaded from jhc.sagepub.com by guest on March 13, 2015

pituitaries rats observed of

the

from described

the in

no

obvious

cycle

except

a

females usually con5). Figure 6 shows a

IMMUNOCYTOCHEMICAL

SPECIFICITY TIM

0.5

z

CHARACTERIZATION

OF TSH

1133

CELLS

TEST

CELLS

0.4

0

z z 4 I-

0.3

0 N

4 02

TTT

0.1

150

UNAS$ORBED

lONG

AMOUNT

FIG. 1. Specmficity tests-Anti-bTSHI1. abolishes staining whereas absorption represent SEM. Number of granule normalized staining intensity is the chromatin is used as background.

ANTIGEN/ML

loG DILUTION

110.000

Absorption with with 100 times more measurements per point following ratio: net optical

ANTIaTSH

1-10 ng of TSH (I-i preparation, NIAMDD) LH or FSH has no significant effect. Error bars is 50-75. As described in a previous paper (13), density of granule/background density. Nuclear

05 3IU,

04

z I-

z

03

z 4 0-

0.2

0

FSH O.I

4

LII!

0 2

UNABSORSED

OPG

IOOPG

AMOUNT

FIG. abolishes staining

2.

Specificity staining significantly,

Effect

ANTIGEN/ML

tests-Anti-rTSH. Absorption whereas 10 pg reduces it significantly. but does not abolish it.

of Fixative

I NG 1:25.000

100 Absorption

with

TABLE

I

TSH

on

Immunoreactivity

scattered

TSH9

mic

Normalized

Fixative#{176}

Staining Intensity’

ONG

DILUTION

pg

cell

SEM

PAP

molecules

0.01

tion

with

±

0.01

stain

varied

PAF

0.18

±

0.007

0.03

There

was

±

0.02

Fixation was for 1 hr at room temperature. bedding was in Araldite 6005. Staining with 1:25,000 anti-rTSH$. o

TSH 1000

from

a

(1-2 times

Em-

preparation, more LH

diestrous

and

NIAMDD) or FSH reduces

female

dilated

that

rough

has

endoplas-

reticulum.

±

p-formaldehyde)

with

The stain for TSHI3 tory or Golgi complex

0.12 0.03

(1.8%

of

IOONG

TSH

granules

1% Glutaraldehyde 2% Glutaraldehyde 4% p-formaldehyde

ANTI-

were rough

observed

near

endoplasmic

in

also

was primarily on secregranules, although a few

intensity

among

an uneven

on some

of the

granules.

weakly,

others

did

To dration

determine and

the heat

Downloaded from jhc.sagepub.com by guest on March 13, 2015

not

or

in associa-

reticulum.

the

distribution Some

stain

granules

The

granules.

of stain stained

at all.

effect of the polymerization

ethanol dehyprocedures

1134

MORIARTY

AND

TOBIN

#{149}0, -

S

s

,#{149}

:#{176}

C

0 5

.

-

4

i

I

I .5

*

S

0

...

4

‘5’.

T

‘4.

#{149}.....

r,.

#{149}

#{149}.

#{149}

t

#{149} #{149}5 ..

#{149}.

4

S.

S

5.

L

#{149},.

‘

....

..

I.

.

Downloaded from jhc.sagepub.com by guest on March 13, 2015

..

IMMUNOCYTOCHEMICAL

CHARACTERIZATION

4,:.

OF TSH

*

#{163}

1135

CELLS

t#{149}

% #{149}. 4 ‘‘

.0 ...

.4,#

#{149}0#{149} 5.

-

#{149}

5

S

-

0

#{149}#{149} #{149}#{149}# #{149}..

0

-

- -

#{149}‘‘2

5#{149}.

S

45.

_________ FIG.

5.

‘LH’

A TSH

cell:

staining

intensity

used

in

6005,

pituitaries

soluble

cases,

the

7);

than

in same

ding,

the

seen

in

stronger

especially

and blocks

in

4%:

stained

was

more

fixed than

FIG. granule.

1:10.0(X)

FIG.

varies

have

anterior

a proceIn

all

TSHI3

been

obtained.

TSH granules postmortem

ing

with

TSHfl in

II).

the

A normal

was

Table

(1:1000

the

stain

same

I).

dilution) cell

of

PAP

removed

after extended in p-formalderesults

with very

weak

stained

type,

appear

unstained.

stain-

(as

we would to

more

although

molecules

in were

granules.

Antiserum

human intensely

there over

was

granules

a of

cells that we have identified as gonadotrophs. Figure 8 shows a TSH cell in the human as well as some cross-reactivity in the nearby gonado-

-‘

Some

fixed

small

Immunocytochemically stained TSH cell (T) from male rat showing stain variation In this cell, granules are arranged peripherally in 1-2 rows along the plasma membrane. anti-rTSH(3. 14.000. 4. Two stained TSH cells (T) containing numerous scattered granules, from an intact

on the granules.

were

specimen specimens

3.

in mtnensmty

from

or anti-rTSHfl, cell

in

x 11,000.

best

anti-bTSHfl

from

monolayer

in human preserved

that surgical When

a

a classical

of the breast; others It was noted that

degraded and/or

and

in

like

the variation

surgically

were intervals,

occurred

predict

similarly

was

looks

note anti-rTSH.

carcinoma at autopsies.

fixation,

stained

cell

Again,

1:10,000

obtained with the 2.5% glutaraldehyde.

However,

embed-

(Table

pituitary

hyde

This

cell.

PAF,

a patient with were obtained

immuno-

in

Fixation,

Araldite

in glutaralde-

in Araldite

in estrous.

in water

tissues.

GMA

p-formaldehyde,

pituitaries

to granule.

granules

TSHI3

Characterization of the TSH cell pituitaries: Relatively few well human

granule

in staining intensity not obvious in the

blocks

embedded

for a TSH

intense

the

following

the

rat

expected

for

embedded

specimens.

was

fixed

by (17).

around

was

a female

range

with

report

araldite

the variation to granule

4, from

the size

from

(GMA)

stain and

in Figure

embedded

type

over

embedded

hyde

and

process

also

cell

diffuse

reactivity

granule

another

however,

that

to cells

are within

methacrylate

Furthermore, from granule GMA

the

were

glycol

more

similar

granules

embedding

described

(Fig.

(T) its

on

the

dure

and

cell

however,

Fixation,

PAF,

Downloaded from jhc.sagepub.com by guest on March 13, 2015

1:10,000

anti-rTSHfl.

granule to Fixation. PAF,

from

male

rat. 10,400.

Stain

1136

MORIARTY

AND

TOBIN

#{149} ;J)%,; Sc

#{149}.

4

44i

.5

4

-. - I

.

‘.5

-44,. S

S

S

,

.5

S

..

‘0.

....

.4 #{149} .

.:

..‘..

8

#{149}.#{149}#{149} #{149}1 S

1 ‘S

#{149}

#{149}8;,_’

#{149}#{149}

I....

I

#{149}

. y

II .4

#{149}#{149}.

‘;. ‘.1b

4.

4.

‘

.

-

45

S

‘5

40

,

-Sr

‘a a

4

4,

a

Io’

..

..-s:,

7

Downloaded from jhc.sagepub.com by guest on March 13, 2015

IMMUNOCYTOCHEMICAL

troph.

The

average

225 nm, diameter.

and they Other

human

thyrotrophs

diameter

CHARACTERIZATION

of the

range in size characteristic

granules

is

from 150-300 features of

include

their

nm the

shape

and

the

presence

that

are

presumed

in Figure

body

1137

CELLS

bodies cell

polyhedral

OF TSH

of

one

or

more

large

to be lysosomes.

8, stain

could

be seen

In the

over

the

lytic

(inset). DISCUSSION

TABLE Effect

of Fixation

Em

bedding

II

and on

Glycol

TSH3

In

Methacrylate

that

immunoreactivity

both

rat

and

stain

for

TSHI3

angular Normalized Staining Intensitv#{176}

Fixative

Staining

with

0.154 0.162 0.2515

1:25,000

and

pituitaries,

the

cells

are

distinguished

by

their

their

content

±

0.01

Our

±

0.009

±

0.02

as small large as

anti-rTSHf3.

measurements

show

#{149}

larger

(1:30

be due

to an

nm).

Some

#{149}. 4

#{149} #{149}

.‘,.

s5

a.

#{149}#{149}

#{149} a

of this

#{149}

.

-

#{149}#{149}*#{149}

#{149}

.

-

‘S

sa

.

.

-

a .‘#{176}.:.ij.’.

4

S.

.

0#{149}

,.

.

--

-

Thyrotroph

from adult human are also seen (arrows).

which

c’5.

:,‘

: #{149}#{149}, #{149} #{149} S.

#{149}

#{149}I

..

. . 5555

W S

.:‘,.!

-

..wS

Fixation.

5’

- .

.4 S

.,‘

.

PU. #{149},

z’

0

Inset is blow-up are 200 A diameter.

!

...

..“

pituitary.

‘

-‘

.., V

(T)

. a

a

.

.

-

.

complexes

.

,.

*

s

#{149}#{149} I

#{149}.

#{149}1

-

8.

4..

..

.

‘5..’.:?o.?.iss,.

in nearby gonadotroph Circles represent PAP (Inset, x:30,600).

, ...

“

S

-

UIc’!...s

S

{.

‘‘‘

S

‘I

:‘

.., #{149}. i

S

1

.

I’

#{149}.

IkI #{149}I1;

.-\...I

4

.5

-

,

of

a

.1-i-I..4

‘T!#{149} ‘.‘

-S

#{149}- -.

.:

-

a

.A-,

.1

-

.

.‘“#{149}

#{149}

could

as a result

#{149} :‘

.‘

#{149}

5*

as is

S

I

#{149}#{149}4NI.. I

#{149}‘#{149} *

‘

5

FIG.

5’

granules

difference

#{149}

11

#{149}.5

..-

#{149}‘#{149}

that

may be diameter

in size

a

.

#{149}

0’

5

8

are

some mean

augmentation

,fl4,

.454 -

-

there

S

S

S

S

that

as 60 nm; however 180 nm, and the

#{149} ..

of granules

are the smallest of all the cell types. According to Costoff, TSH granule size (in rat pituitaries) ranges from 40-150 nm with a mean of 80 nm.

SEM

1% Glutaraldehyde PAF (1.8% p-formaldehyde) 4% p-formaldehyde

shape

human

3.5;

of lytic Fixation

glutaraldehyde. Cross-reactive granules body which shows some reaction of TSHfl. PAF, 1:1000 anti-human TSHI3. 12,000

FIG. 6. TSI-I cells (T) may also have fewer granules scattered among rough endoplasmic reticulum. This cell is from a diestrous female rat. Stain varies from granule to granule. No stain was evident in the rough endoplasmic reticulum of this cell. Fixation, PAF 1:10,000 anti-rTSHII. x6,975. FIG. 7. Immunocytochemically stained TSH cell (T) from tissue fixed in 2% glutaraldehyde and embedded in glycol methacrylate. There is some variation in staining from granule to granule as well as more intense staining. Fixation and embedding is less than adequate (note holes); however, with recent improvements we find GMA to be a reasonable alternative. Fixation, PAF 1:25,000 anti-rTSH. x7,000.

Downloaded from jhc.sagepub.com by guest on March 13, 2015

1138

MORIARTY

the stain. However, our measurements with those of Farquhar (maximal ter-150-200

nm) report angular

distinguish

from

the

its

angular

by

granules

220

studies in the

pointed out that cells are difficult

LH

cell

which

shape

LH

average

cells;

demonstrates nm or more.

a

distinguishing

content

diameter

check

that Thus,

(9,

of

there cells

their

10).

Our

are that

cells look

granule

there are no granule size

marker

of

size

granules remains

between

of 200 a good

the

two

cell

populations. Furthermore, with the light his

our studies are microscopic studies

colleagues

presence

(1-3)

of

LHfl

that

and

in agreement of Baker and

demonstrated

TSHL

in

two

to the

were

entire

LH

separate

cells,

the

granules

diameter) along the TSH

are are

not

not

periphery. rows.

as

TSH

and

in the

the

lie

in

7.

or

Kawarai

Y,

lysosomes.

in rat

TSH

artifactual

K, Oota

a state

10. Moriarty

GC:

our

of crinophagy Perhaps

weak

structures

are

show

or lytic

this surgical

staining

was

that

digestion

occurring

removal

13.

of gonadotroph

anti-hTSH9

sera

the

ence tion

of some cross-reacting antibodies. tests with this antiserum are in

The

specificity

of

the

reaction

in

the

79:808,

and

by

electron

1966

microscopic

immunocyto-

97:1215. NS:

complex.

a

1975

Electron

adrenocorticotropin antibody and

GC,

microscopic

producing cell soluble peroxidase-

J Histochem

Cytochem

Moriarty

15.

CM,

Sternberger

LA:

immunocytochem istry with unlaand the peroxidase-antiperoxia technique more sensitive than J Histochem Cytochem

1973

stimulating

Absorphuman

1972 Moriarty

21:825,

pres-

progress.

ante-

of rat pituitary gonadotroph: morphology and cytochemistry

Halmi

14. Moriarty GC, nocytochemical

granules

indicates

revealed

Endocrinology

GC,

Ultrastructural beled antibodies dase complex: radioimmunoassay.

of pitui-

the

in

20:590, 1972 12. Moriarty GC, Halmi NS: Adrenocorticotropin production by the intermediate lobe of the rat pituitary. An electron microscopic-immunocytochemial study. Z Zellforsch Mikrosk Anat 132:1,

in the

of the

Electron

antiperoxidase

on the presumed It may reflect studies

Moriarty

Cytochem

Adenohypophysis: ultrastructural a review. J Histochem Cytochem

study of the with unlabeled

glycoprotein hormones, TSH readily diffusable. It may also

interval between tary and fixation. the

Such

as

tissue

gonadectomized

Endocrinology

GC:

chemical studies sex difference in

bodies

of the

rats

cytochemistry, 21:855, 1973

11.

of

peroxidase-

Y: Corticotrophs

glands

Endocri-

Localization

1970 pituitary

thyroid-

study.

18:161,

microscopy.

cells.

since

the three the most stores.

large

of the stain 8 is uncertain.

diffusion,

among appears

contain

PK:

Soc

alterations

following

microscopic

Nakane

Mem

Cytologic

gland

on the ultrathin sections with antibody method. J Histochem

9. Moriarty

between human the cells are more cells are polyhe-

gland.

JF:

pituitary

thyroidectomized

more

pituitary

antigen labeled

rior

smaller

two

the fully

CITED

anterior

an electron 55:857, 1954

8. Kurosumi

at the

with were

studies.

19:79, 1971 MG, Rinehart

anterior

ectomy: nology

average

distributed

may

cells

The significance lysosome in Figure

The

stellate,

they

resemble

reflect

nm

of the

Endocrinol 6. Farquhar

of LH cells.

found

TSH

(220

are several differences TSH cells. In the rat whereas human TSH

Human

that

larger

as regularly

Also,

There and rat stellate, dral.

are

can be confused with ACTH cell. In ACTH

staining which

1. Baker BL: Studies on hormone localization with emphasis on the hypophysis. J Histochem Cytochem 18:1, 1970 2. Baker BL, Pierce JG, Cornell JS: The utility of antiserums to subunits of TSH and LH for immunocytochemical staining of the rat hypophysis. Am J Anat 135:251, 1972 3. Baker BL, Yu YY: The thyrotropic cell of the rat hypophysis as studied with peroxidase-labeled antibody. Am J Anat 131:55, 1971 4. Costoff A: Ultrastructure of Rat Adenohypophysis, Academic Press, New York, 1973 5. Farquhar MG: Processing of secretory products

molecules

and distributed in a single row evenly plasma membrane (9, 11, 12, 16).

cells

granules

with

TSH

these

LITERATURE

used.

Another cell type that stellate TSH cells is the

not

and

in

by cells

the

cells. This is also in agreement with electron microscopic immunocytochemical studies of Nakane and his colleagues (15, 7) in which antisera

proved by its and anti-TSH/3,

well to

is distin-

and

have shown that although population of stained TSH

like

is

characterized

it was TSH

nm

TOBIN

TSH cell anti-bTSHfl

do agree iiame-

(5).

In another granulated, guished

AND

Tobin RB: Ultrastructural immulocalization of anti-thyroid hormone

(fITSH)

binding

sites

in

human pituitary adenoma and in pituitaries from normal and thyroidectomized rats. Proc 57th Ann Mtg Endocrine Soc 96:154, 1975 Nakane PK: Application of peroxidase-labeled antibodies

to

Downloaded from jhc.sagepub.com by guest on March 13, 2015

the

intracellular

localization

of

IMMUNOCYTOCHEMICAL hormones. 1971

Acta

Endocrinol

Suppl

(Kbh)

CHARACTERIZATION

1970 17.

Spaur

RC,

Garner

Improvements as an embedding

LL,

of glycol medium

Hon

CA,

Moriarty

methacrylate and for immunocytochemi-

cal studies. 1975, p 30

153: 190,

16. Siperstein ER, Miller KJ: Further cytophysiologic evidence for the cells that produce adrenocorticotrophic hormone. Endocrinology 86:451, GC: its

use

OF TSH

18.

Proc

Sternberger

HG:

The

LA,

CELLS

26th Hardy

unlabeled

Ann PH

antibody

1139 Mtg Jr,

Histochem Cuculis

enzyme

JJ,

Soc. Meyer

method

of

immunohistochemistry: preparation and properties of soluble antigen-antibody complex (horseradish peroxidase-antiperoxidase) and its use in identification of spirochetes. J Histochem Cyto-

chem

18:315,

1970

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