Vol. 47, No. 1
Printed in U.S.A.
T H E AMERICAN JOURNAL OP CLINICAL PATHOLOGY
Copyright © 1967 by The Williams & Wilkins Co.
STAINING FOR NERVE FIBER AND CHOLINESTERASE ACTIVITY
IN FRESH FROZEN SECTIONS
TATSUJI NAMBA, M.D., P H . D . , TOSHIO NAKAMURA, M.D., P H . D . , AND DAVID
GROB, M.D.
Department of Medicine, Maimonides Hospital, and Slate University of New York,
Downstate Medical Center, Brooklyn, New York
The staining of muscle for both nerve
fibers and cholinesterase lias been described
in several reports,2""4'6' u - 1 2 but only with
tissues fixed by formalin before sectioning,
which inactivates some enzymes. This report
describes a procedure for staining nerve
axons or myelin sheaths and cholinesterase
in sections from unfixed frozen muscle and
other tissues that has resulted in improved
demonstration of these structures.
cyanide solution (0.25 Gm. in 100 ml. of
distilled water) for 10 min. at room temperature.
4. Rinse twice with distilled water, for 5
min. each time.
5. Place in absolute ethanol for 1 hr.
6. Rinse with distilled water for 1 min.
7. Place the section in silver nitrate solution for 90 min. at 37 C. (10.0 Gm. AgN0 3 ,
0.05 Gm. CuS0 4 -5H 2 0, and 0.1 Gm. CaC0 3 ,
in 100 ml. of distilled water).
S. Rinse with distilled water for 5 sec.
9. Immerse the section for 10 min. in the
reducing solution (1.0 Gm. of hydroquinone
and 10.0 Gm. of Na 2 S0 3 , in 100 ml. of distilled water), with gentle shaking during the
first minute.
10. Wash twice in distilled water for 5
min. each time.
11. Dehydrate in 90% ethanol and then
in absolute ethanol; clear in xylene, and
mount in balsam.
METHODS
Preparation of Tissue for Staining
1. Freeze unfixed fresh tissue in dry iceisopentane for approximately 30 sec.
2. Section the tissue at 30 to 50 n with a
microtome in a cryostat at —16 to —IS C.
The tissue may be sectioned at different
thicknesses and used for other staining procedures, including demonstration of enzymes that are inhibited by formalin fixation.
3. Place the tissue section on a coverslip
and thaw it in formalin vapor at room temperature.
Combination Staining of Nerve Axons and
Cholinesterase
Add the following procedure for staining
of cholinesterase between steps 2 and 3 of the
procedure for staining of nerve axons:
1. Incubate the section for 60 min. at 37
C. or at room temperature in the modified
acetylthiocholine solution of Koelle and
Friedenwald8 (S ml. of supernatant from a
mixture of 7.8 ml. of aqueous solution of
0.15 Gm. of acetylthiocholine iodide and 2.6
ml. of aqueous solution of 0.06 Gm. CuSOr
5 H 2 0), and in 92.0 ml. of 0.05 M Veronalacetate buffer, pH 6.1 or 4.9, which contains
0.075 Gm. of glycine and 0.05 Gm. of
CuS0 4 -5H 2 0. The incubation is carried
out at pH 6.1 and 37 C. for tissues with low
cholinesterase activity, and at pH 4.9 and
room temperature for tissues with high
cholinesterase activity, such as the motor
endplate.
2. Rinse with distilled water for 30 sec.
Staining of Nerve Axons
1. Fix the sections for 1 hr. at 4 C. in
buffered formol-calcium with magnesium
and cadmium (10.0 ml. formalin, 1.0 Gm.
CaCl2, 0.5 Gm. MgCl 2 -6 H 2 0, and 0.1 Gm.
CdCl 2 -23^H 2 0, in 100.0 ml. of 0.07 M.
Veronal-acetate buffer, pH 6.45).
2. Place in distilled water for at least 1 hr.
at 4 C. The sections may be kept in distilled
water for as long as 7 days, if this is desired.
3. Immerse the section in potassium ferriReceived, April 27, 19GG.
Dr. Nakamura's present address is Department
of Anatomy, Kanazawa University School of
Medicine, Kanazawa City, Japan.
This study has been supported by United States
Public Health Service Grant No. NB-03464 from
the National Institute of Neurological Diseases
and Blindness.
74
Jan.1967
STAINING FOR NERVE AND CHOLINESTERASE
75
FIG. 1 (upper, left). Nerve endings, including the annulospiral ending of the muscle spindle in the
tibialis anterior muscle of the rat, demonstrated by silver deposit. After fixation with formol-calcium
this muscle section was stored for 7 days in distilled water without untoward effect on staining. Axon
stain. X 420.
FIG. 2 (upper, right). Fine autonomic nerve plexus in the iris of a rat, demonstrated by silver deposit.
Axon stain. X 420.
FIG. 3 (middle, left). Motor endplates and motor nerve terminals in the levator palpebral muscle
of a human being. The nerve axons and cholinesterase activity at the motor endplate are visualized by
deposits of silver. Cholinesterase and axon stain. X SO.
FIG. 4 (middle, right). Motor endplates and motor nerve terminals in the tibialis anterior muscle of
the rat. The nerve axons and cholinesterase activity at the motor endplates are visualized by deposits
of silver. Cholinesterase and axon stain. X 410.
FIG. 5 (lower, left). Motor nerve terminals and motor endplates in the intercostal muscle of a patient
with myasthenia gravis. The larger arrow indicates a branch of the axon terminal accompanied by
cholinesterase active structure, the subneural apparatus; the small arrow points to a nerve branch without cholinesterase active structure. Cholinesterase and axon stain. X 320.
FIG. 0 (lower, right). Cross-section of myelinated nerve fibers in the tibialis anterior muscle of a rat
stained by acid hematein. Myelin stain. X 320.
76
NAMBA ET
Staining of Myelin Sheath
1. Fix the tissue section for 1 hr. at 4 C.
in glutaraldehyde solution (12 ml. of 25%
glutaraldehyde, 0.45 Gm. of NaCl, 0.3 Gm.
of CaCl2, 0.25 Gm. of CdCl 2 -2K H 2 0, in
distilled water, total volume 100 ml.).
2. Wash twice in distilled water for 5 min.
each time. The sections may be kept in distilled water for as long as 3 days, if this is
desired.
3. Immerse for 1 hr. at 60 C. in dichromate solution (5.0 Gm. of K2Cr207 and 1.3
Gm. of CaCl2, in 100 ml. of distilled water).
4. Rinse twice with distilled water for 1
min.
5. Stain for 3 hr, at 60 C. in hemotoxylin
solution (0.1 Gm. of hematoxylin, 0.02 Gm.
of NaI0 3 , in 100 ml. of distilled water; prepare fresh by mixing for 1 hr. with a magnetic stirrer).
6. Wash in distilled water for 5 min.
7. Immerse at 37 C. in differentiating
solution (0.25 Gm. of K 3 Fe(CN) 6 , 0.25 Gm.
of Na 2 B 4 O 7 -10 H 2 0, in 100 ml. of distilled
water) until myelin sheaths are clearly distinguished. This usually requires IS to 4S hr.
S. Wash in distilled water for 10 min.
9. Dehydrate in 90% ethanol and then in
absolute ethanol; clear in xylene, and mount
in balsam.
Combination Staining of Myelin Sheath and
Cholinesterase
Add the following procedure between
steps 2 and 3 of the procedure for the staining of the myelin sheath.
1. Incubate the section in modified acetylthiocholine solution, as described previously.
2. Wash for 10 min. in distilled water.
3. Place for 5 min. in ammonium sulfide
solution (5.0 ml. in 95 ml. of distilled water).
4. Wash for 5 min. in distilled water.
RESULTS
The methods described in this communication were applied to skeletal muscle,
smooth muscle, blood vessels, skin, iris,
lacrimal glands, optic nerves, and sensory
and autonomic ganglia of man, rats, and
mice, as well as to skeletal muscle of dogs
and rabbits. The nerve axons, myelin
Vol. 47
AL.
sheaths, and cholinesterase activity were
clearly visualized in these tissues.
The method for staining of nerve axons
clearly identified the nerve axon of sensory
nerves (Fig. 1), autonomic nerves (Fig. 2),
and motor nerves (Figs. 3 to 5), as well as
nerve cell bodies of ganglia. These structures were stained by silver deposits, producing a brown-black or black color easily
differentiated from the background. The
method employed is a modification of Honjin's method, 6 which is based on Cajal's
silver method. After fixation, sections could
be preserved in distilled water at 4 C. for as
long as 7 days, if this was desired (Fig. 2).
Combined staining of axons and cholinesterase resulted in clear delineation of
these structures in the same section. The
product of cholinesterase activity, thiocholine, was first visualized as red deposits by
potassium ferricyanide treatment. 7 These
were replaced by black deposits of silver
after silver staining (Figs. 3 to 5). The silver
stain of the nerve axon was also considerably increased in intensity by this procedure.
The method for staining the myelin
sheath of peripheral nerves clearly identified
this structure, which stained a dark blue
color (Fig. 6). The method employed was a,
modification of Baker's acid hematein
method.1 Fixation of tissue sections of glutaraldehyde10 yielded better results than
fixation by formalin or formol-calcium, and
the sections could be stored in distilled water
at 4 C. for 3 days without deterioration.
Combination staining of myelin and cholinesterase resulted in clear delineation of
these structures in the same section, the
myelin sheath1 staining dark blue and cholinesterase activity reddish-brown.
DISCUSSION
The methods for staining described in this
paper have resulted in a clearer demonstration of axons or myelin and cholinesterase
activity in the same section. The methods,
which are simple and reproducible, have
facilitated study of these structures and of
synapses in skeletal muscle and other tissues.
Studies on the distribution of motor endplates and the relation of nerve fibers to the
subneural apparatus in the extraocular mus-
Jan. 1967
STAINING FOR NERVE AND CHOLINESTERASE
cles have been reported separately.9 Combination staining for axons and cholinesterase of muscle from patients with myasthenia
gravis has demonstrated that elongated,
branched axon terminals are not consistently
associated with the subneural apparatus
(Fig. 5), whereas staining for cholinesterase
alone has not demonstrated such variety.2
The use of unfixed frozen tissue instead of
formalin-fixed tissue permits the use of sections for other staining methods, including
methods for enzymes that are inactivated by
formalin fixation, and staining with hematoxylin and eosin. This is especially helpful
when only a small sample of tissue is available, as in needle biopsy specimens.
SUMMARY
Methods of staining axons and myelin
sheaths, and combination staining of axons
or myelin and cholinesterase, in sections
from unfixed frozen tissue have been described and have been applied to skeletal
muscle, smooth muscle, blood vessels, skin,
iris, lacrimal glands, optic nerves, and sensory and autonomic ganglia. A modification
of the silver method was utilized for staining
axons, a modification of Baker's acid hematein technic for staining myelin, and a modification of the acetylthiocholine method for
localizing cholinesterase activity. Thepresent
methods, which are reproducible and relatively simple, permit sections of the same
specimen to be used for other staining technics, including those that require fresh
frozen sections. Combination staining of
cholinesterase and nerve terminals is believed to have wide application in studies of
the structure of synapses, especially the
neuromuscular junction.
77
Acknowledgments.
D r . Abraham R. Kantrowitz
and the staff of the D e p a r t m e n t of Pathology
supplied most of the human material used in this
study.
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