the use of formalin fixation in an improved method for ... - SAGE Journals

128

TO THE

LETTERS

THE

USE

OF

FORMALIN

FIXATION

IMMUNOFLUORESCENCE

EDITOR

IN

AN

IMPROVED

HISTOCHEMICAL

NEUROPHYSIN

IN

THE

antirabbit

‘y-globin

ocyanate

(Burroughs

sectioning

phosphate

buffer

a cryostat.

Recently,

this

been applied to the demonstration the hypothalamoneurohypophysial (2),

rat

(6)

and

guinea

pig

technique

has

of neurophysin

system

(1).

in

of the

In a recent

pig

publication

buffer

K430

complex, accessory sony paraventnicular

supraoptic

the

occur

on a Dry

a such

found

an

sodium

and

to

Hope

(2),

fresh-frozen

Bouin’s

tissue

fluid

cent

did

results.

many

There

proteins

tissues

in

proteins

are

pituitary blocks.

After ded

fonmalin

slides, were

were

phosphate

chloride,

“sandwich”

7.6)

g disodium

water.

The

technique (6) work

(3)

serum, was

using

supported

1:4

The

sections

washed

by

a grant

New Zealand Medical Research Council Auckland Medical Research Foundation.

1.

this

the

can

cell

be

the

seen.

in

ical

in

cryostat

presence (Fig.

of proc1, b and

technique

the to

to stain

either

d).

applying for

aldehyde

for-

neuro-

fuchsin

or

-

progress,

of formalin

hypothalamus

the

After

it is possible by using

was use

from

bodies

h istochem

demonstration on

bodies

in

fixed

Zimmerman and

Bouin’s

of neurophysin by

an

(7)

et al. fixation in the

prior monkey

immunoperoxidase

in a and liter

using

neu-

from

and

3.

the

phosphate

The the

Evans JJ, Watkins WB: physin in the neurosecretory thalamus and neurohypophysis osmotically stimulated strated by immunofluorescence

Localisation of neuroelements of the hypoof the normal and guinea-pig as demonhistochemical

techniques. Z Zellforsch Mikrosk Anat 145:39, 2. Livett BG, Uttenthal LO, Hope DB: Localization

g sodium

antiovine with

postfixed tissue

REFERENCES

at

glass

dihydrate/1 rabbit

by immuno-

tissue from

revealed the

-hematoxylin

the

for-

cut

phosphate

diluted

to

dehy-

were

stained

While

embed-

of 8.5

were

which

method.

in

was

hydrogen

sections

and from

material -alum

hor-

periods

consisting phosphate

a on

from

observed

of frozen cell

sections

secretory

and that

albumized and

film

The

nuclei

malin-fixed chrom

that

and

sections

using

recorded

made.

on sections

sharp

reported

in

block

to

consistently

or

growth

in xylene

for

et al. (4)

long

tissue

to water

dihydrogen

rophysin-III ‘This

(pH

and

statement

in xylene.

ethanol

buffer

1.28

0.16 g sodium distilled

the

in

sections

Pasteels

microscope were

seen

immunofluorescent is

in formalized

for

attachment

through

immunohisto-

studying

cleared

deparaffinized

taken

cell

immunofluorescence

fixation,

after

Often

appreciation

(5).

at 60#{176}C.Paraffin

and,

the

immunofluores-

stored

alcohol,

in paraffin

8-10

emanating

Uttenthal

Pearse’s

when

by tissues

through

esses

saline-formalin

exception

results

malized

in

antigenicity to

the

prolactin

paraffin drated

their

contradiction

and

either

were

as observed

sections appeared diffuse and lacked fine detail (Fig. 1, a and c). The images obtained from the corresponding saline-formalin paraffin-embedded sections were

We

of cryostat

stained was

previously slices

washed

This blocks

finding

satisfactory

alcohol

9 g

Livett,

is now a growing

excellent

mone

give

retain

such reported

in

not

92%

groups using

The

dried,

and

Ektachrome were

in 30%

cryostat.

slide,

Images

prints

with

reactant.

soaked

a

fluorescent

speed

cell

containing

This

fixation

the

fluorescence

was on

7.6)

filter.

white

hypothalami

in the

by

that

Laborlux

high and

block

Immunofluorescence

secondary

black

tissue

1:10

second

saline-formalin.

increases

slice.

reported

found

a Leitz

as the

to a glass

sheep isothi-

diluted

7.6)

(pH

and

fluorescein

may

snap-frozen.

of the

detail

tissue

that

who

large

dramatically

histologic

suffers

formation.

formalin

ml

either

damage

when are

fixation

treated

contrast

crystal

hypothalami

chloride/l0()

chemically

tissue

common

of 4

into

mixture

that

ice

initial

solution

resolution

of

of

as

that

aqueous

using

reactant to

sectioned

buffer

sections.

Kodak

acces-

to contain

plunging

Ice-acetone

is especially

tissue

have

by

in

result

phenomenon of

the

found

fixed

attached

phosphate

first

Wellcome),

and

were

All

disadvantage

as

and

were

of tissues

nitrogen

from

supraoptic

the conjugated

the

paraffin

antigen.

freezing

liquid

the

nucleus

nucleus

neurophysin-like

The

including

as

(pH

frozen

sections

addition,

groups

7.6)

Occasionally,

in

cells

(pH

sucrose,

we showed neurophysin to be localized in the penkaryon of cells of the magnocellular paraventnicular and supraoptic nuclei of the guinea pig (1). In other

FOR OF

HYPOTHALAMUS’

Im munofluorescence histochemistry is now widely used for the detection of trace amounts of tissue antigens. In general, tissues are snap-frozen prior to on

METHOD

INVESTIGATION

4.

1973 of

neurophysin-Il in the hypothalamo-neurohypophysial system of the pig by immunofluorescence histochemistry. Philos Trans R Soc Lond [Biol Sci] 261:371, 1971 Nairn RC: Fluorescent Protein Tracing. Ed 3. Livingston, London, 1969 Pasteels JL, Gausett P, Danguy A, Ectors F, Nicoll CB, Varavudhi T: Morphology of the lactotropes and somatotropes of man and rhesus monkeys. J Clin Endocrinol Metab 34:959, 1972

5. Pearse plied.

AGE: Histochemistry, J and A Churchill, Ltd,

Theoretical London,

and 1961,

App 146

LETTERS

FIG.

guinea

1. a, immunofluorescence

pig after

snap-freezing

localization

rabbit

antiovine

neurophysin-Ill

and

THE

129

EDITOR

in cells adjacent to the third ventricle of a normal Ice-acetone mixture. b, neurophysin-containing cells This figure represents a transverse section cut from a saline-formalinProcesses can be seen emanating from a few cell bodies. c. diffuse nucleus of the fresh-frozen guinea pig hypothalamus after staining with

of the tissue

close to the guinea pig third ventricle. fixed paraffin-embedded hypothalamus. images of cells in the paraventnicular

TO

sheep

specific immunofluorescence of neurophysin saline-formalin prior to paraffin embedding.

of neurophysin

block

in Dry

antirabbit

‘y-globulin

coupled

in the guinea pig paraventnicular Processes and cell nuclei are

to fluorescein nucleus. The visible. . 120.

isothiocyanate. tissue

was

d, fixed

in

130

LETTERS

6. Watkins physin

WB, in the

tem of the cross-species

Evans

JJ:

Demonstration

of neuro-

hypothalamo-neurohypophysial

normal and reactive

dehydrated rat anti-neurophysins.

TO

sysby the

use of Z Zell-

forsch Mickrosk Anat 131:149, 1972 7. Zimmerman EA, Hsu KC, Robinson AG, Carmel PW, Frantz AG, Tannerbaum M: Studies of neurophysin secreting neurons with immunoperioxidase techniques employing antibody to bovine neuro-

THE

EDITOR

physin.

I. Light

and bovine August

microscopic

tissues.

14, 1973

I

Endocrinology

findings

in monkey

92:931,

1972

W. B. WATKINS J. J. EvAI’Js Postgraduate School of Obstetrics and Gynaecology University of Auckland Auckland, New Zealand