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Luxol fast blue staining of degenerating myelinated fibers.

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Luxol Fast Blue Staining of Degenerating
Myelinated Fibers
ANN B. SNODGRESS, CHARLES H. DORSEY AND LEONARD B. LACEYJ
Physiology Division, Naval Medical Research Institute, Bethesda, Maryland
Since the development by Kluver and
Barrera ('53) of a method of myelin sheath
staining using an alcoholic solution of an
amine salt of sulphonated copper phthalocyanine which is available as Luxol fast
blue MBS or, more recently, MBSN (du
Pont'), other investigators of the histological uses of this dye (Pearse, '55; Margolis and Pickett, '56; Becker and Treurnich, '59) have continued to employ it in
alcoholic solution according to the basic
procedure of Kluver and Barrera. However, several substitutions have been suggested for the cresyl violet counterstain
originally recommended.
Kluver and Barrera ('54) observed that
in chromated material their technic also
stains astrocyte fibers and Pearse ('57)
has stated that Luxol fast blue will stain
mitochondria "suitably fixed (presumably
by a chrome salt fixative). Shanklin and
Nassar ('59) reported the use of Regaud's
fixative as a basis for their combined alcoholic Luxol fast blue-periodic
acidSchiff stain for the mitochondria of renal
epithelium. Formalin, however, has remained the fixative of choice for the staining of normal myelin sheaths. Margolis
and Pickett observed that alcoholic Luxol
fast blue produces a "weaker and muddy
myelin stain following chromation."
While exploring the use of Weigert's
primary mordant on formalin-fixed tissue
for iron-hematoxylin staining of mitochondria at neural endfeet as recommended by Armstrong, Richardson and
Young ('56), and the earlier use of prolonged chromation by Smith and Mair
('09) for staining degenerating myelin in
frozen sections, i t occurred to us that
Luxol fast blue might be substituted for
Kultschitsky's hematoxylin which has been
used by these authors. However, alcoholic
Luxol fast blue, while staining mitochon-
dria, lacked the intensity of color that is
expected from this stain. Both normal and
degenerating fibers were only faintly
stained.
With the idea that an aqueous solution
might be a satisfactory vector for Luxol
fast blue in staining heavily chromated
material, an aniline water Luxol fast blue
has been used. By increasing the concentration of aniline beyond the cold water
saturation point at which Altmann used
it in preparing aniline acid fuchsin, we
have obtained a stable aniline Luxol fast
blue which, when used on chromated nervous tissue, is not only an intense mitochondrial stain but also a stain for degenerating myelinated fibers both peripherally
and within large fiber tracts of the central
nervous system. Normal myelinated fibers
stain, but, in general, with less intensity
and with less variability than degenerating
ones. Macrophages and their inclusion
droplets are stained both in the central
nervous system and in degenerating peripheral nerves. Astrocyte fibers and
sucker feet, ependymal cell processes, the
blepharoplasts of ciliated ependyma and
erythrocyte envelopes also stain. Adipose
tissue, where present adjacent to peripheral nerves, stains a pale blue or bluish
green. Through counterstaining, neuronal
and axonal cytoplasm, oligodendroglia and
Schwann cells are demonstrated.
MATERIAL
For testing the staining procedure with
aniline water Luxol fast blue, we have
used the cat as a source of mammalian
material and the chicken for avian mate1 Present
address: Department of Anatomy,
University of Kentucky College of Medicine, Lexington, Kentucky.
2 Samples of Luxol fast blue (MBS and MBSN)
were supplied to us through the courtesy of E. I.
du Pont de Nemours and Company.
83
84
ANN B.
SNODGRESS,
CHARLES H. DORSEY AND LEONARD B. LACEY
rial. Cats I and 11, in which the right eye
had been enucleated, were sacrificed on
the 4th and 21st postoperative day, respectively. Cat 111, in which a portion of the
left optic tract was destroyed by an electrode lesion ( 3 ma X 60 sec. ), was killed
after an interval of 14 days. Cat IV (figs.
1-3) which received multiple bilateral
damage to dorsal spinal nerve roots, primarily at cervical and upper thoracic
levels, as a result of convulsive seizures
during exposure to high pressure oxygen,
was sacrificed on the 12th day following
injury. These animals were anesthetized
with nembutal and the brain perfused
in situ with 10% formalin containing 2%
crystalline sodium acetate. The brains
were subsequently stored for one month
(and in the case of Cat IV for 6 months)
in isotonic sodium acetate formalin. Two
cat sciatic nerves (figs. 4, 5 ) which had
been severed 14 days and 19 days previously, together with the contralateral normal sciatic were fixed in sodium acetate
formalin. Chicken sciatic nerves (figs. 6,
7 ) obtained from animals paralyzed as a
result of oral dosages of a triaryl phosphate compound and their normal controls
were fixed in either phosphate-buffered
10% formalin or in 10% formalin containing magnesium carbonate.
PROCEDURE
I. Mordanting
Solution A (Weigert’s primary mordant)
Potassium dichromate
Chromium fluoride
Distilled water
5 gm
2 gm
100 ml
Dissolve with a little heat; cool and filter.
Solution B
Potassium dichromate
Distilled water
5 gm
100 ml
( a ) Blot lightly formalin-fixed tissue
and place in solution A at room temperature for 5 to 7 days. The volume of solution should be at least 20 times the volume
of tissue. Slices of brain or spinal cord
( 3 to 10 mm thick) or entire segments of
nerve may be used.
( b ) Transfer to solution B at 37°C for
one to two weeks, changing the solution
every third day.
In order to determine optimal chromation, it is advisable to process rapidly a
small portion of the tissue after 5 days
chromation in solution A. Seven days in
this solution alone has been adequate for
chicken sciatic nerve. Satisfactory chromation for cat sciatic nerve and central
nervous system was obtained with additional chromation for one week in solution
B. In overchromated tissue both normal
and degenerating myelin sheaths are refractory to Luxol fast blue and axons tend
to stain eccentrically blue-green.
2. Embeddina
( a ) Wash in running tap water (12 to
24 hours).
( b ) Dehydrate and clear through:
( 1) 80% alcohol (one to two
hours),
( 2 ) 95% alcohol (two changes in
two to three hours),
( 3 ) Terpineol (two changes in 12
to 24 hours),
( 4 ) Xylene (two changes in 30
minutes).
( c ) Infiltrate with paraffin M.P. 5658°C ( 4 changes in 4 to 6 hours), embed
and block.
3 . Section preparation
( a ) Sectioning of this brittle material
is facilitated by inverting the unshaven
surface of the paraffin blocks overnight in
a shallow pan containing equal parts o€
glycerin and water.
( b ) In cutting, the microtome knife is
ice-chilled but the paraffin blocks are allowed to remain at room temperature.
( c ) Prior to ribboning, the cut surface
of the block is moistened by the application of wet absorbent cotton for two to
three minutes.
( d ) Sections, cut at 5 CI or not thicker
than 8 u, are floated on water maintained
at 4 7-50 C .
( e ) After deceration with xylene, the
slides are transferred to absolute alcohol
and immersed in 0.5% celloidin in etheralcohol. Shake off excess celloidin, allowing the remaining thin film to become firm
but not dry. Place slides in 75% alcohol
for 5 to 10 minutes before transferring to
distilled water .
O
85
LUXOL FAST BLUE STAINING
4 . Staining method
(a) Pretreatment
1. Place in 0.5% potassium permanganate for 5 minutes.
2. Rinse briefly in distilled water.
3 . Transfer to 2% oxalic acid for three
minutes.
4. Wash in running tap water 10-15
minutes.
5. Immerse in distilled water.
The potassium permanganate - oxalic
acid bath may be omitted but sections are
differentiated more evenly and easily after
its use.
(b) Staining
Place overnight at 50°C in Luxol fast
blue prepared as follows:
Distilled water
Aniline oil (reagent grade)
Glacial acetic acid
Luxol fast blue
1000 ml
30 ml
10 ml
1 gm
The solution is allowed to stand for
three days at 57°C with occasional shaking. In practice we prepare the solution in
a glass-stoppered bottle and store it in an
oven. Solutions held in hot storage as long
as two months have remained satisfactory.
The hot stain should be filtered through
rapid filter paper before using.
( c ) Differentiation
1. Allow slides to cool to room temperature.
2. Transfer to 70% alcohol briefly to
remove excess stain.
3. As in the original method of Kluver
and Barrera, immerse briefly in 0.05%
lithium carbonate and continue differentiation in 70% alcohol, repeating these two
steps with fresh solutions until satisfactory differentiation is achieved. Mitochondria will appear blue-green against an
almost colorless background. Normal myelinated fibers will be stained, but degenerating ones will show a more intense bluegreen color. If the sections are overdifferentiated, they may be restained by repeating step (b).
(d) Countemtaining
1. Rinse in distilled water.
2. Stain in cresyl violet three minutes.
To prepare this solution we use:
Kresylechtviolett (Bayer )
4 gm
Distilled water heated to 80°C 380 ml
Allow to cool, filter and add:
M / 5 acetic acid
M/5 sodium acetate
15 ml
4 ml
3 . Rinse in distilled water.
4. Immerse in 80% alcohol briefly.
5. Transfer to 95% alcohol containing
three drops of glacial acetic acid per 100
ml for 15 seconds.
6. Complete differentiation in two
changes of 95% alcohol of approximately
one minute each. Unlike the original
Kluver method in which the formalin-fixed
tissue makes possible a brilliant stain for
Nissl substance, cresyl violet, when used
on chromated tissue, functions only to
convert the initial blue-green of the Luxol
fast blue to a deeper purplish blue. Differentiation should be continued until the
neuronal cytoplasm is colorless.
7. To remove the celloidin film, at this
point transfer the slides to absolute alcohol
for three minutes and follow with immersion in acetone for 5 minutes. Rinse
briefly in absolute alcohol and return to
95% alcohol. Decelloidinizing may be
omitted if difficulty is experienced in
maintaining the section on the slide.
8. Stain in erythrosin for 15 seconds.
For this we use:
Erythrosin (Chroma)
95% alcohol
0.5 gm
100 ml
9. Differentiate in two changes of 95%
alcohol until cytoplasm is pink. If, at this
stage, difficulty is encountered, it is most
frequently because of insufficient diff erentiation of the preceding cresyl violet which
renders the tissue refractory to erythrosin. This may be corrected by repeating steps ( 5 ) , (6) and (8). Occasionally,
it may be desirable to correct under-differentiation of the Luxol fast blue. This may
be done by downgrading the sections
through 70% alcohol to 0.05% lithium
carbonate and returning them through
70% and 95% alcohol to erythrosin without restaining in cresyl violet.
10. Unless the celloidin film is preserved, complete dehydration in absolute
alcohol, clear in xylene and mount in a
synthetic resin. To maintain an intact
celloidin film n-butyl alcohol may be substituted for absolute alcohol in dehydration. Sections prepared by this method
have retained their original color over a
period of two years.
86
A N N B. SNODGRESS, CHARLES H. DORSEY AND LEONARD B . LACEY
RESULTS
Erythrosin as a cytoplasmic counterstain produces a pink background for the
blue mitochondria and myelin sheaths.
It is a useful stain for the demonstration
of axonal cytoplasm. On longitudinal section, the normal axon presents a pink,
homogeneous, smooth appearance bearing
along its length thin, rodlike mitochondria
which in transverse section stipple the
axon. In a peripheral nerve or spinal root,
it is surrounded by a pale blue, somewhat
dense myelinated sheath with the neurokeratin network staining more deeply than
the myelin component. On transverse section, however, it is more difficult to distinguish between neurokeratin and myelin.
Within the central nervous system axonal
staining is similar to the peripheral picture, but normal myelin, particularly of
large axons, may appear only a faintly
greenish yellow in contrast with deep blue
neurokeratin. This is equally true of transverse and longitudinal sections.
The degenerating myelinated fiber of
large or medium size presents a variable
picture. Axoplasm, where it remains, appears coarsely granular, tortuous, fragmented and more deeply stained. Mitochondria may be rounded and swollen or
else absent. Occasionally droplets of blue
staining material are seen within the rose
to violet axoplasmic fragments. These
axoplasmic fragments are often visible
within the distorted and degenerating
myelin sheaths, even when sheath disintegration has proceeded to the droplike
stage. In the droplike stage, the degenerating sheath substances may be globular
or lobate, homogeneous or else somewhat
reticulated and suggestive of neurokeratin,
with a color range from yellow-green to
deep blue.
On cross section, degenerating fibers
are frequently seen which have lost the
erythrosinophilic axon core and, instead,
stain entirely blue with a laminated appearance.
DISCUSSION
Among the defects of this method we
would list its tendency to stain normal
neurokeratin more deeply than normal
myelin. This may be particularly evident
when a longitudinal section of a fiber is
made through the sheath but lateral to the
axon. However, the paler myelin is often
visible within the interstices of the neurokeratin.
A more serious limitation to the use of
this method results from its failure to
stain degenerating preterminal fibers in
sufficient quantity when compared with
the Nauta (’57) method of silver impregnation of degenerating axons in the central nervous system.
From Cat IV we were able to compare Nauta preparations from a segment of upper thoracic cord with sections
taken from an adjacent segment and
stained with Luxol fast blue. Although
fibers in cross section in the cuneate
fasciculi were somewhat difficult to interpret in the Nauta sections, degenerating fibers coursing through the dorsal
horns were observed in droplike disintegration. Many fine degenerating preterminals were seen in Clarke’s columns and
the zonae intermediae, and a lesser number in the ventral horns. Both dorsal roots
at this level, which were stained in Luxol
fast blue, contain degenerating fibers. In
contrast with the Nauta silver impregnations, in cord sections stained with aniline
Luxol fast blue, degenerating fibers observed in cross section in the dorsal funiculus are easy to distinguish from normal
ones, but degenerating fibers traversing
the dorsal horn, while visible, are few in
number. Although in direct microscopic
observation with a X 40 objective it was
possible to discern droplets of what appeared to be blue staining lipid material
in Clarke’s columns and the zonae intermediae, the amount of this material was
too small to photograph. It may be assumed that the inadequacy of this stain in
demonstrating degenerating fibers within
cord gray matter is due in part to the presence of fewer degenerating fibers in these
5-u sections than in the 25-cl sections used
in the Nauta preparations. Furthermore,
the color density of the stain results in a
poor visual contrast between the droplets
of fragmented material actually present
and the abundance of normal axons, dendritic processes and deeply staining mitochondria. There is, however, the possibility that there is some loss of lipid breakdown material in the dehydrating or paraffin embedding stage of treatment despite
87
LUXOL F A S T BLUE STAINING
the prior long chromation period to which
it is subjected, or that the aniline water
Luxol fast blue is less sensitive for fine degenerating axons and their preterminal
arborizations than is the Laidlaw ammoniacal silver carbonate solution used in
the Nauta procedure.
For neuroanatomical studies the Nauta
silver impregnation method has the great
merit of affording high contrast between
degenerating preterminals in droplike disintegration and a background in which
normal axons are largely suppressed. Consequently, in sections prepared by the
Nauta technic, areas containing degenerating preterminals may be effectively
mapped and photographed in black and
white. Except when used on peripheral
nerves or on major tracts in the central
nervous system, in its present stage of
development, the aniline Luxol fast blue
procedure is difficult to photograph except
in color.
However, when used on sections of peripheral nerves, aniline Luxol fast blue
with a n erythrosin counterstain offers
the advantage of a good contrast both between the normal and degenerating myelin sheaths and between normal and degenerating axon cores. The mitochondrial pattern within the axon and within
Schwann cells and macrophages is also
demonstrated by this method.
CONCLUSION
A method of using a n aniline water solution of Luxol fast blue for the demon-
stration of degenerating myelinated fibers
and mitochondria on postchromed formalin-fixed tissue has been presented.
LITERATURE CITED
Armstrong, J., K. C. Richardson and J. 2. Young
1956 Staining neural end feet and mitochondria after postchroming and carbowax embedding. Stain Technol., 31: 263-270.
Becker, B. J. P., and D. S. F. Treurnich 1959
Luxol fast blue as a stain for Mallory’s alcoholic hyaline. Ibid., 34: 261-263.
Kluver, H., and E. Barrera 1953 A method for
the combined staining of cells and fibers in
the nervous system. J. Neuropath. Exp.
Neurol., 12: 400-403.
1954 On the use of azaporphin derivatives (phthalocyanines ) i n staining nervous
tissue. J. Psychol., 37: 199-223.
Margolis, G . , and J. P. Pickett 1956 New applications of the Luxol fast blue myelin stain.
Lab. Invest., 5: 459-474.
Nauta, W. J. H. 1957 A method of silver impregnation of degenerating axons in the central nervous system. WRAIR-19-57, Research
Report, Walter Reed Army Institute of Research, Washington, D. C.
Pearse, A. G . E. 1955 Copper phthalocyanins
as phospholipid stains. J. Path. Bact., 70:
554-557.
1957 The chemistry of copper phthalocyanin staining for phospholipids and its application to the lipidoses. In: Cerebral Lipidoses, A Symposium. J. J. Cumniings, ed.
Charles C Thomas, Springfield, Ill., p. 99.
Shanklin, W. M., and T. K. Nassar 1959 Luxol
fast blue combined with the periodic acidSchiff procedure for cytological staining of
kidney. Stain Technol., 34: 257-260.
Smith, J. L., and W. Mair 1909 A n investigation of the principles underlying Weigert’s
method of staining medullated nerve. J. Path.
Bact., 13: 14-27.
PLATE 1
EXPLANATION O F FIGURES
All figures in this plate are from coronal sections through the medulla oblongata of Cat IV.
1
Lateral portion of cuneate fasciculus. Normal fibers have pink axon cores surrounded
by pale blue sheaths. Degenerating fibers stain varying shades of blue without differentiation of axonal material. X 760.
2
Lateral cuneate nucleus. Numerous degenerating fibers without discernible axon cores
are present. X 760.
3
Cuneate nucleus. Only normal fibers are seen. X 760.
LUXOL FAST BLUE STAINING
Ann B. Snodgress, Charles H. Dorsey and Leonard B. Lacey
PLATE 1
89
LUXOL FAST BLUE STAINING
Ann B. Snodgress, Charles H. Dorsey and Leonard B. Lacey
4
Normal sciatic nerve (cat).
PLATE 2
x 760.
5
Degenerating sciatic nerve (cat, distal portion 14 days subsequent to section).
6
Normal sciatic nerve (chicken). X 760.
x 760.
7 Degenerating sciatic nerve (chicken, 15 days subsequent to an oral dosage of a triaryl
phosphate compound). X 760.
90
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