1-1018$02.O0/0 OF HISTOCHEMISTRY

0022-1554/78/261 THE JOURNAL

Copyright

© 1978 by The

AND

Histochemical

CYTOCHEMISTRY

Society,

Vol. 26, No. 11, PP. 1018-1020,

Inc.

Printed

Brief

PERMANENT

Reports

FLUORESCENT STAINING IN ISOLATED CELLS’ GEORGE

Physics Received

Department,

for publication

OF

NUCLEIC

ACIDS

I. MALININ

Georgetown July

1978

in U.S.A.

University,

19, 1978, and in revised

Washington, form

D.C.

August

20057

11, 1978 (BR

78- 122)

Staining of fixed cells, blood smears and chromosomes with 0.1% w/v of 3, 5, 7, 2’, 4’pentahydroxyflavanol (morin dehydrate) in 70% ethanol after brief mordanting with 5% w/v aluminum ammonium sulfate results in permanent fluorescence of cellular nucleic acids. Incubation in ribonuclease solution before mordanting, or 5-mm hydrolysis with INHCL at 60#{176}C selectively abolishes RNA fluorescence, while the incubation in deoxyribonuclease solution abolishes DNA fluorescence. The morin-mordant complex bound to nucleic acids is stable to photodegradation and up to fIve years of storage. The

advent

tological sustained odology surprising

of automation

for

the

quantitative

H.

cy-

Cultured

analysis of dispersed cells has generated a effort to refme further cytochemical methavailable for this purpose. Thus, it is not that owing to high quantum yields and

specificity

of

staining

certain

methods

tometry

fluorochromes,

have

and

image

been

the

adapted

analysis

for

of

chemical

seems,

agents

therefore,

method

and that

obviating

useful

even

specific these

to and

2. The

dium

simple

storage.

restrictions

3.

would

It

serve

199(8)

Cell

monolayers

a

solution 10 mm

neutral

purpose.

for

in dispersed

permanent fluorescent (DNA) and ribonucleic

staining of (RNA) acids

and

is, therefore,

in this

kept

cells

presented

munication.

4. MATERIALS

AND

METHODS

1. Five

percent

monium 2.

Morn

w/v

sulfate, dehydrate

filtered

solution

aqueous

Grand

aluminum

am-

as

w/v

pH 3.1. (Aldrich)

0.1%

in 70% ethanol.

Ribonuclease tion

in 0.2

(Sigma) M phosphate

5. Deoxyribonuclease 0.2 M phosphate This Naval

investigation Research

Contract

as 0.5

j.tg/ml

buffer

at pH

(Sigma) solution buffer at pH 6.5.

was supported

by

the

Office

fluid

and

fixed

absolute

cells

ethanol for

fixed

subse-

cells

were

fixative.

Island,

were SA

N.Y.), calf.

cultured

medium

for (Gibco,

supplemented serum The

then

incubated

for

of0.1 ment

tg/ml was

of colcemid. carried out

0.075

M KCL

10 min

6.5

were

respectively,

ethanol

formol

in McCoy’s

dispersed

solu-

in

v/v

balanced

Carnoy’s

in 75%

The

15% v/v fetal phytohemagglutinin.

3. 0.1 N HCL. 4.

hr

Earl’s

fixed,

lymphocytes

65-70

I. Reagents:

then

twice

in the

Human

on coverslips

The

kept

use.

10%

24 hr in Carnoy’s

washed

were in me-

antibiotics.

cold

and and

were

quent

com-

with

formol.

were

A method deoxyribonucleic

10%

cultures

and

grown

twice

for

with antibiotics.

with

glutamine

salt

es-

on coverslips

supplemented

serum,

in

minimal and

skin

explants

calf washed

fluorescent

glutamine human

from

monolayers

Eagle’s

supplemented

serum,

primary

grown

cellular

a simple

calf

as

using

medium

w/v

systems (1, 3-5). Unfortunately, however, one of the major drawbacks of numerous fluorescing dyes is their well known lability to rapid photodegradation, effects

grown

tubes

sential

cytopho-

of disassociated

were

Leighton

fluorescent flow

cells:

1 . L-929

two

at room

suspensions

with

and 2.5 cultures

hr in the

.tg/ml were

presence

Hypotonic treatfor 10 mm with temperature. were

then

The fixed

for

acetic acid mix-

in 3:1 methanol

ture.

in HI.

Human tionally, for

of

N00014-78-C-0320.

the

Carnoy’s 1018

blood smears were air-dried briefly and subsequent fluid

use or methanol

prepared then

or fixed for

conveneither

additionally 10 mm.

stored

in

BRIEF Iv.

sections were prepared as usual using Carnoy and formol fixed material. Staining procedure: After testing a number of variants, the following procedure has been evolved for staining of DNA and RNA in cul-

Formol

Tissues

V.

tured

cells,

A.

blood

smears

and

chromosomes.

(1) Cell monolayers, blood smears and chromosome spreads were hydrated with distilled water and immersed for 5 mm (chromosomes, 30 min) into aluminum ammonium sulfate (2)

were

washed

37#{176}C or alternatively

with

dis-

essed Microscopy.

D.

hydrolized

for

5 mm in 1.0 N HCL at 60#{176}C. They are then washed in distilled water and processed further as stated. staining. Cells are incubated for 1 hr at 37#{176}Cin

DNase

solution

and

proc-

as described.

Stained

preparations

served

with

scope

equipped

citation filters.

Leitz

were

Orthoplan with

and

an

K530

BG32

and

ob-

MicroUG1

ex-

510 barrier

somes not

was

sharply

fluorescent

morn

after

Carnoy

somes

was

mordanting

with

conditions. show any diation

cells

aluminum

stained alum

with

exhibited

ponents.

Excellent

nor

chelation

ble effect

with

respect

conventional

and

mid-bodies

technique. inclusions

were

Nucleoli, as well

easily

nuclear

membranes,

as intercellular

demonstrable

bridges

(Figs.

1 and

3).

After degradation with RNase or acid hydrolysis the staining of RNA was totally abolished (Fig. 2) while the intensity of DNA fluorescence was not impaired. No fluorescence could be elicited in cells sequentially incubated in DNase and RNase solutions. Staining of cells at controlled pH only with morn resulted in weak diffuse fluorescence.

under

of chromostated

staining

with

EDTA

to staining

had

any

detecta-

specificity.

Under

stated

staining

conditions,

the

localization

of

nucleic acids in cultured cells appears to be very high as evidenced by the results after degradation with the nucleases. This selectivity for instance affords excellent

localization

of DNA-containing

cellular

inclusions

present in cell cultures infected by DNA viruses (7), and ofintercellular bridges and mid-bodies seen during delayed cytokinesis (6, 9). As was the case with cell cultures, chromosome may

be

banding

mined. Stained also

remarkable quenching

rocytes

photographed

these can

of

to iron,

were

obtained

with

iron

results be

for remains

demonstrated

fluorescent to

be

deter-

the exception of erythromorphological detail and photodegradation. The complex by erythrocytes since

analogous

with

cultured

containing

suggest

subsequent

permanent

however,

blood cells, with exhibited sharp stability to of the fluorescent

attributed

mordanted terms,

readily

Feasibility

be

cytoplasmic

resolution

A method for permanent fluorescent staining of nucleic acids in isolated cells and chromosomes is based on the formation of an exceptionally stable complex of morn with an aluminum salt (2) which in its turn is selectively bound to cellular nucleic acids.

results

any

were

DISCUSSION

tive

with

erythrocytes conditions.

obtained

nucleases

may

favorably

The staining

sections: Stained tissue section exhibited nonspecific fluorescence of all tissue comNeither the preliminary degradation with the

extremely

compared

ex-

However,

or storage.

cytes,

and

likewise

fluorescence.

(Fig. 4) the intense fluorescence did not tendency to fade following prolonged irra-

Tissue an intense

very intense greenish-yellow fluorescence which showed no visually detectable decrease in intensity irrespective of the duration of illumination or storage (Fig. 1). The resolution of cytological structures was sharp

any

invariably

karyotyping.

fixed

as described

stable

delineated. under

chromosome

cultures:

stained and

unlike Carnoy fixed cells, the nuclear staining was more diffuse and the contrast of chromatin and nucleoli against the less intensely fluorescent background was not as sharp. Blood films: Very intense fluorescence was exhibited by the white blood cells and platelets. In the case of female blood, the “drumstick” of XX chromo-

spreads RESULTS

Cell

cells

intense

Chromosomes:

then

tilled water. (3) Washed cells were stained with morn for 5 miii, rinsed again with distilled water, dehydrated in ethanol, cleared in xylene and mounted in resin. B. DNA staining. Before mordanting with aluminum alum, the cells are incubated in the RNase solution for 1 hr at

C. RNA

fixed

hibited

solution.

They

1019

REPORTS

that

salts.

and cells

In

no fluorescent in

normal

negabriefly

practical erythperipheral

blood. In contrast to isolated cells, tissue sections failed to exhibit any specific fluorescence, although an intense general fluorescence was readily induced. A possibility

that

the

non-specific

fluorescence

may

be

due to the interfering divalent cations was explored by protracted chelation with EDTA, but no substantial improvement of staining specificity was detected. High specificity and the stability of the fluorescent morn-

BRIEF

1020

REPORTS

FIG. 1. L-929 cells stained for RNA and DNA. Note intercellular bridge between two cells. Microphotography performed 2 yr after staining and after at least 5 hr exposure to UV, x1500. FIG. 2. Primary skin culture stained for DNA after RNA degradation. Photographed 5 years after staining. Cumulative exposure to UV at least 6 hours, x1500. FIG. 3. L-929 cells in delayed cytokinesis. Stained for DNA and RNA. Diffuse fluorescence of the cell is due to the difference between focal planes of the two cells and intercellular bridge containing a prominent midbody. One year old preparation, x1500. FIG. 4. Human chromosomes stained for DNA and RNA six months before photography and exposed for at least 3 hr to UV, x1500.

aluminum method tative yet

complex affords localization

an

mordant

that

and

acids

possibility nucleic

acids

gests that the quantitative nations of cellular nucleic

the

reliable

of nucleic

unproven to

indicates

a simple

that may

for

in isolated binding

be

described

means

quali-

cells.

As

of aluminum

stoichiometric

sug-

cytofluoremetric determiacids may be feasible.

indebted

to

Dr.

G.

5, Clay

Cameron BF, Bobbit banas A: A procedure

S, Cayer

Bahr

for

reviewing

the

J Histochem SB, Paoletti

1. B#{246}hm N, Sprenger E: Fluorescence cytophotometry: A valuable method for quantitative determination of nuclear Feulgen DNA. Histochemistry 16:100, 1968 2. Feigl F: Spot Tests. Elsevier, N. Y. 1958, p. 182 3. Goerttler K, Ehemann V, Tschahargane C, Stoehr

8. Morgan animal synthetic 1950

9. Mullins kinesis

TJ,

and

JM,

nucleic

observations

transformed Inst 43:693,

Morton

HT,

Biesele cells.

TT:

hamster 1969

Parker

cells in tissue culture. medium. Proc Soc

in D-98S

D,

1977 complex

acids.

J

MM and Malinin TI: DNAand intercellular bridges in Hopkins Med J 122:42, 1968

GI: Cytochemical

enovirus type-12 cells. J Natl Cancer

M, Ingram

Cytochem 25:525, C: A fluorescent

6. Malinin GI, Vincent containing mid-bodies tissue culture. Johns

7. Malinin CITED

acid analCytochem

D, Gaddis R, Lief SB, Cafor dissociating Ayre scrap

between ethidium bromide Mol Biol 27:87, 1967

manuscript.

LITERATURE

and deoxyribonucleic cell nuclei. J Histochem

25:560, 1977 4. Leif RC, Nordquist

samples. 5. Lepecq

ACKNOWLEDGMENTS I am

M: Monodispersal ysis of prostatic

on the adembryo

RC: Nutrition I. Initial Exp Biol

Terminal J Cell Biol

phase 83:672,

of studies of Med 73:1, of cyto1977