194

Histochemistry
A Significance

of Protein

Groups

of Alloxan- and Ninhydrin-Schiff

Methods for Protein

Yasuo DEGUCHI
Departmentof Oral Surgery,OsakaUniversityDental School,Osaka.
Introduction
The
most

alloxan-

specific

reaction5),
method7)
also
fibers,

the
and

being

and

and

ninhydrin-Schiff

available

reactions

dinitrofluorobenzene
the

employed

amyloid,10-12)

methods
in protein
H-acid

method,4,6)

dimethylaminobenzaldehyde
in histochemical
etc.

have

detections

been

accepted

histochemistry.1-4)
the

reaction
of proteins,

coupled

to be the
The

for tryptophane8,9)
elastic

millon

tetrazonium
are

and kollagen

195

The

results

in the present

study

indicate

that

comparatively

similar

findings

are observed
in the determination
of proteins
methods except for the dimethylaminobenzaldehyde

in tissue sections with the above

reaction,
and that the con-

tent

always

of

amino

acid

methods.13)
The main
and

in tissue

purpose

of this

ninhydrin-Schiff

effects

of some

work

methods

acetic

acid

cannot

was to confirm

for protein

histochemical

on

and trypsin,
of terminal

The

tissues

at

the

the

of

materials

embbeded

16.

liver,

protein

these

of the alloxan-

to clarify

the

in reference

possible

to digesting

kidney,

salivary

for

cold

histochemical
to

being

affected

absolute

glands,

microtome

be

very

acetone

or

of

useful

by

to

fixation
in

tissue

preserve

or

10 %

oral

sections

demonstration

believed

without
in

pancreas,

prepared

the

were

fixed

Methods

were

For

conditions

it

embedding.

formalin,

then

paraffin.

frozen

ethanol

rat

sections

were

in

and

by

and also to study the changes

of the
amino
groups
with
nitric
acid and

and

intestine

8, or

natural

Fresh

small

raw-frozen

relatively

Some

the

and

thickness

protein,
in

including

stomach

estimated

etc.

Meterials

mucosae,

be

the specifity

groups,

fixatives

effects of crystalline
pepsin
stainability
after
blocking
anhydrous

protein

and

sections

dried

methanol,

trichloracetic

in

10 %

acid

room

temperature,

formalin,

alcohol

for

fixed

Zenker's

15

minutes,

in

solution,
were

acetone,

absolute

Bouin's

employed

solution,

in

the

or

following

method.
Alloxanwith

and

1 %

alloxan

Ninhydrin

water

in

then

solution,

and

mounted

with

Millon

were

and

running

more

than

immersed

three

changes

After

were

2-3

treated

hours

at

deamination
in

rinsed

were

water.

for

oxidative

were

in

sections

ethanol,

in

but

ethanol

Schiff's

reagent

10 %

dehydration

sodium
the

37.
amino

no

absolute

in
of

of

color
and
for

bisulfide

sections

were

balsam.
: Millon's

modified

the

water.

solution5)

method.

with
at

water

fixed

ethanol

by

sections

rinsed

in

absolute

in

The

The

base

soluble

discolored,

washed

treated

in

very

in

Schiff

immediately

Reaction

developed

ninhydrin

solution.

completely

minutes,

Bensley's

Methods1):

produce

were

water

untill

0.5

alloxan

Alloxan

developed

and

or

and

groups.

30

Ninhydrin-Schiff

was

Non-fixed

Millon's

sections

solution

positive

for

portion.

Glycerol

prepared

mounting

after

3-5

Then

according

hours

they

preserved

dried
at

were
in

in

to
room

Gersh
temperature

37.

brownish

washed

in

1 %

reaction

for

and

several

color

nitric

acid

months.

Observation
Liver
the
a

: The

presence
more

recognized

cytoplasm

of

granular
in

protein

of

cells

substance

reaction
the

hepatic

intercellular

than

in
in
space.

was

great
frozen
Similar

well

stained

amount.
sections.
staining

diffusely,

Paraffin
Granular
reaction

indicating

sections
reaction
were

showed
was
observed

196

between the central and peripheral parts of hepatic lobules. The nuclei were
moderately reactive in the formalin fixed tissues, while they were more intensely
stained than the cytoplasm in the paraffin sections.
Kidney : Epithelial cells of the distal and medial convolutions were stained
markedly by alloxan Schiff and Millon's reactions. Erythrocytes
in the glomerulus and tubules were also stained pink with the alloxan-Schiff
method. The
cortex of the kidney showed a strong stainability.
Pancreas : The cytoplasm of pancreatic acinar cells was strongly reactive,
and the cells of Langhans islets were weaker. Sometimes, some pancreatic
acini were stained more intensely than the other cells subjected to the DMABnitrite reaction.
It was assumed that those findings might be related with
the production of proteolytic pre-enzymes,
trypsinogen
and chymotrypsinogen
in the pancreatic acini.
Salivary gland : In the submandibular
gland, the serous cell was moderately
reactive and the duct cell strongly
reactive.
Nuclear staining of the acinar
and duct cells was moderate
in the formalin fixed sections and less in the
acetone fixed ones. The mucous acini were scarcely
reactive.
The mucous
acini of the sublingual gland were not or slightly reactive as the mucoid cells
in the submandibular
gland.
The basal portions of the acinar and demilune
cells were stained moderately, and the duct cells showed a good stainability.
Mucous cells were not stained by the alloxan-Schiff
and Millon reactions.
Parotid gland ; Both the acini and duct cells showed a more intense reaction
than that of the serous acini of the submandibular
gland.
The stainability
was similar to that in the cytoplasm of the pancreas.
Stomach : The gastric epithelium was generally moderately
stainable and
the chief cells of the gastric gland stained
more intensely.
The muscular
zone of the stomach showed also a strong stainability.
Intestines : The intestinal
epithelium
was moderately
stained, but the
mucous intestinal gland in the duodenum and bowels showed a negative reaction.
Blocking
The
and

blocking

simple
Frozen

with

for

out,

with
pH

block

reduced
pically.
prolonged

to

with

N/10

sulfhydryl

these

in

a slight

also

the

benzoylation,

chemical

or

10

30

with

time

was

were

immersed

NaOH,

for
of
the

in

coupled

M/10

hours.

well

used.

acetic

anhydride

benzoyl

chloride

alcohol

with

at

37.

The

monoiode
This

has

Danielli.6)

Deamination
in

pyridine

in

pyridine

M/100
other

acetic

agent

sufficient

to

NaOH
sections

acid,

adjusted

been

known

to

protein.14-16)

which

nuclear

90

hours

in
20

alloxan-

tetrazonium
and

2-3

were

5 %
in

is

according

formalin

with

benzoylation

agents

specificity,

dinitrofluorobenzene

groups

degree

by

acetylation

pretreatments,

But

acetone

Incubation

10 96 formalin

to
active

By

and

groups

histochemical

24 hours,

with

performed.

fixed

end

fixed

pretreatment

were

the

acid

carried

and

protein

demonstrating

sections

nitrous

were

of

for

Test

the

and
reactions
reactions

staining

ninhydrin-Schiff
could
were
became

not

reactions
be

not

detected
reduced

stronger.3,4,17,)

were
macrosco-

even

by

197

Digestion

with Crystalline

Pepsin

and Trypsin

Digestion by purified peptic enzymes may be one of the accepted methods

for a histochemical
detection of protein.4,13) Actions of pepsin and trypsin are
suggested by the study of Mazia and Kaufmann et al. on the protein structure
of chromosomes.18,19) They believed that main actions of trypsin
were attributable to the digestion of peptic links in basic proteins.
Mazia found that
pepsin would not digest histones in solution but would digest acidic protein.
Kaufmann
also believed that the principal action of pepsin is removal of
tryptophan containing proteins of the non-histone
variety.
It is well known
that pepsin has a marked digestive effect on native collagen fibers.
Acetone and 10 % formalin fixed paraffin sections of major salivary glands
were utilized in this test.
A solution containing
enzymes was prepared as
follows ; 10 mg of crystalline
pepsin was added to 10 ml of N/10 HCl, and 10 mg
of phosphate buffer (pH 6.8). Deparaffinized sections by xylene were incubated
for 30-90 minutes in peptic solution, and 60-120 minutes in tryptic solution.
The finding obtained with a mild peptic and tryptic digestion showed no
structural
impairment
of the sections, and a prolonged digestion caused a
structural
destruction.
By peptic digestion for 90 minutes, the duct cells and
submandibular
acini virtually loosed its stainability,
only the cell membrane
and intercellular
fibers were stained ; so the digesting
action of pepsin was
higher than that of trypsin.
Furthermore,
the stainability
of the sections
became weaker after both digestions.
Discussion
Alloxan
fixed

and

at

this

the

reaction,

when

the

generally

ninhydrin

terminal
free

are

coupled

tetrazonium

method

tyrosine

It

is

an

low
is

or

acid

The
But

the

interesting
is

from

protein

result

method

polypeptide
for

that

of

protein

DMAB-nitrite

the

reactivity
with

not
of

the

Table

1.

Effects

of Reactivity

be

tissue

histochemical

loyed.4,23)

by Fixatives

is
in

been

alloxan-Schiff
; and

demonstrate

obtained
could

has

chains

method

results

of

histochemical

the

method

are

dissolved

It

these

and

acid

be

groups.
for

groups

the

related

protein

the

may

groups,

components
that

closely

process

DNFB-H

tryptophane.

in

which

In

responsible

amino

and

groups

chains.1,4,13,20-22)
molecular

groups

terminal

oxidize -amino

employed

protein

method

tryptophane.13,21)

alloxan-Schiff

to

protein

terminal -amino

likewise

amino

the

the

terminal

of

and

that

mostly

demonstrates

kind

acid

histochemical

method

known
of

amino

considered

methods

are
portion

said

this

the

mainly
to
study,

detect
the

defined.
sections
fixatives

with

the
emp-

198

This
result
will be related
with a problem
of the fixing
mechanism
histochemical
fixatives
on tissue.
But, the chemical
and physical
actions
fixatives
state

on tissue
that

of methylene

one

proteins

of the

bridge

have

chemical

on amino

or

not been

made

reactions

of formaldehyde

sulfhydryl

clear

groups,

today.
and

Many
is the

mercurial

authors
formation
fixatives

of
of

199

are

combined

carboxyl

with

acid

groups

Tissues

fixed

in 80 % alcohol

erence in their stainability

fixed in absolute
alcohol,
denaturating

agent

sulfhydryl
This
tions

of proteins,

especially

with

hydroxyl

and

groups.
containg

of protein

trichloracetic

difference
chains.

in position

The

results

proteolytic
method.

and

A comparative
and

the

The

result

will

between
fixation

between

amino

blocking

test

seemed
studies

quantitative

to intensify

acid

of the

enzymes

acid

showed

no diff-

in alloxan-Schiff
method comparing
with the sections
although
trichloracetic
acid is known to be a strong

groups.14,16,21)
difference
of reactivity

after

trichloracetic

to

alloxan-Schiff

and

sulfhydryl

of protein

of the

protein-bound
sulfhydryl

to depend
groups

groups

the

of

and

be considered

realize

estimation

reaction

may

on the reactivity

be published

the

and

excellence

in the

total

protein

the

protein

of the digestion

of the

by

alloxan-Schiff

in the histochemical

in another

reac-

upon

protein

in tissue

mothod

is carrying

on.

paper.

Summary

The
in

alloxan-

and

reference

to

chemical

method

1)
tissue,

Generally,

observed

distributions

previous

positive

groups

were

availability

for

studied
the

histo-

and

positive

of

hydrolytic

observed
results

stomach,

reactions

many

the

the

in

considerable

protein

enzymes

the

obtained

the

liver,

coincidence

groups

which

muscular

in

and

were

was

the

histoche-

obtained

in

the

studies.
2)

In

fresh

frozen

formalin

fixation,

Paraffin

embedded
3)

acinar

By

of

the

sections,

whereas

peptic

cells

maining

the

cell

the

no

sections

also

digestion

for

sublingual

90

or

1.

Alloxan-Schiff
Nuclear

Fig.

2.

Fig.

3.

reaction.
reaction

is

Alloxan-Schiff
Nucleus

reaction.
is

stained

Alloxan-Schiff

more

cytoplasma

of

fixed

10%

fixed

in

10%

fixation.

stainning.
of

the

scarcely

duct

and

stained,

re-

stained.

epithelium. ~
hornified

fixed

than

Acetone

were

after

acetone

Plate

oral
and

formalin

intensely

reaction.

the

still

observed

after

nuclear

glands

border

negative

were
seen

marked

parotid

cell

or

was

minutes,

Acetone

slight

reaction

stain

showed

and

membrane

nuclear

nuclear

Explanation

Fig.

protein

the

were

From

gland

of

for
and

reactions

fibers.

salivary
the

tissue
sections.

kollagen

between

mical

in

raw-frozen

marked
and

pancreas,

methods

localization

with

elastic

kidney,

ninhydrin-Schiff

their

oral
Fig.

200

layer

is

dissolved

epithelium. ~

little.

200

1.

sublingual

and

submandibular

salivary

gland. ~200
Fig.

Fig.

4.

5.

Sublingual

and

lylation

for

Nucleus

is

Alloxan-Schiff

submandibular
hours.

stained

gland.

Coupled

tetrazonium

reaction

after

A.
strikingly.

reaction.

Formalin

fixed

stomach

epithelium. ~

400

benzo-

200
Fig.

6.

Alloxan-Schiff
fling

Fig.

7.

is

Alloxan-Schiff

reaction.
clearly

observed. ~
reactions.

Liver,

formalin

fixed,

paraffin

section.

Nuclear

stain-

200
Liver,

formalin

fixed,

fresh

frozen

section. ~200

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