Chapter XXI: ). Imbed in celloidin preferably. When decalcification

has been carried out place sections in an alkaline solution before
staining, and stain for a longer period than usual. Methylene-blue
and eosin stain osteoblasts and osteoclasts very well. Van
Gieson’s is an especially useful stain for ordinary work; osteoid
tissue is red, calcified areas yellow. Sections of bone without
decalcification can be prepared by fixing, hardening and staining
in bulk; the bone is then sawn in the dried condition, and the
sections ground down to the required thickness. _Schmorl’s_ methods
for the preparation of bone-sections have practically superseded
all other staining methods.

Schmorl’s Thionin-picric-acid Method.

1. Fix in formol or formol-Müller’s preferably.

2. Decalcify in formol-nitric acid or Ebner’s alcoholic HCl acid
solution.

3. Wash thoroughly in water. After-harden in increasing strengths
of alcohol; freeze or imbed in celloidin (not paraffin); cut.

4. Transfer sections to water for 10 minutes.

5. Stain sections, well spread out, for 5-10 minutes in a
solution of 2 cc. of a saturated solution of thionin in 50 per
cent alcohol and 10 cc. of water, to which 1-2 drops of ammonia
are added.

6. Wash in water.

7. Stain ½-1 minute in hot saturated and cold filtered
watery-solution of picric acid.

8. Wash in water.

9. Differentiate in 70 per cent alcohol until the color ceases to
come away in blue-green clouds, 5-10 minutes or more.

10. Dehydrate in 96 per cent alcohol.

11. Clear in phenol xylol; xylol; balsam.

Lacunæ and canaliculi dark brown to black; bone cells red;
ground substance yellow or brownish yellow. Calcified areas take
a darker yellow than non-calcified. This method consists in an
impregnation with a fine precipitate rather than a staining. Should
the precipitate be too heavy in portions of the section it may be
removed by thorough washing between 9 and 10.

Schmorl’s Thionin and Phosphotungstic or Phosphomolybdic Acid
Method.

1. Fix thin pieces of fresh bone in formol, then in Müller’s for
6-8 weeks, or 3-4 weeks in the incubator. Fixation is best at
37°C.

2. Decalcify in Ebner’s hydrochloric acid solution. Wash
thoroughly. After-harden in alcohol. Imbed in celloidin or
paraffin. Cut very thin sections.

3. Water for 10 minutes.

4. Stain in the alkaline-thionin solution, as in previous method,
for 3 minutes.

5. Wash in water.

6. With glass needles transfer sections to a saturated watery
solution of phosphotungstic or phosphomolybdic acid for a few
seconds.

7. Wash 5-10 minutes, until section is sky-blue in color.

8. Fix the stain for 3-5 minutes in ammonia 1 part, water 10
parts.

9. Transfer directly to 90 per cent alcohol; change twice.

10. Dehydrate; clear in carbol-xylol; balsam.

If the ground-substance is too dark differentiate in acid alcohol
before dehydrating, and then wash thoroughly before beginning the
dehydration.

Outlines of lacunæ and canaliculi are dark blue; ground-substance a
light or greenish blue; cellular elements a diffuse blue. Schmorl
advises this method for growing bone; in rickets the well-ossified
areas alone stain well. Both of the Schmorl methods can be used for
the study of =teeth= as well.

Staining of Sharpey’s Fibres (v. Kölliker).

1. Harden, decalcify, imbed, cut.

2. Place section in strong acetic acid until it becomes
transparent.

3. Stain in a saturated watery solution of indigo carmine for
15-16 seconds.

4. Wash in water; mount in glycerin.

Fibres red; ground-substance blue.

III. CARTILAGE.

Cartilage stains deeply with hæmatoxylin; with Weigert’s fibrin
stain it holds the blue; with thionin and polychrome methylene-blue
stains for mucin cartilage stains metachromatically red.

IV. CONNECTIVE TISSUES.

_a._ =Connective-tissue Fibrils.= The demonstration of
connective-tissue fibrillæ or reticulum is of great importance in
the differential diagnosis of sarcoma and carcinoma. _Van Gieson’s_
method is the best stain for the coarser fibrils, but does not
bring out the finer reticulum as well as Mallory’s aniline-blue
method.

1. =Mallory’s Reticulum Stain.=

1. Fix in mercuric chloride or Zenker’s. After-harden in alcohol;
imbed in celloidin or paraffin; cut.

2. Stain in 1/10 aqueous acid fuchsin 5-10 minutes.

3. Transfer directly to the following solution and stain for 20
minutes or longer:—

Aniline-blue, water-soluble (Grübler) 0.5 grm.
Orange G (Grübler) 2.0 grms.
1 per cent aqueous solution of phosphomolybdic acid 100 cc.

4. Wash and dehydrate in several changes of 95 per cent alcohol;
dry with absorbent paper.

5. Clear in xylol or origanum oil.

6. Mount in balsam.

Fibrils and reticulum of connective tissue, amyloid, mucin
and connective-tissue hyalin are blue; nuclei, protoplasm,
fibroglia fibres, axis cylinders, neuroglia fibres and fibrin
are red; elastic fibres are pink or yellow; red blood-cells and
myelin-sheaths are yellow.

2. =Mall’s Method for Reticulum.=

1. Digest frozen sections of fresh tissue for 24 hours in a
solution of Parke, Davis and Co.’s pancreatin 5 grms., soda
bicarbonate 10 grms., water 100 cc.

2. Wash carefully in water.

3. Place section in test-tube half-full of water and shake
thoroughly to remove cells.

4. Spread out on slide and allow to dry.

5. Allow a few drops of the following solution to dry on
slide:—picric acid 10 grms., absolute alcohol 33 cc., water 300
cc.

6. Stain for 30 minutes in the following mixture: acid fuchsin 10
grms., absolute alcohol 33 cc., water 66 cc.

7. Wash in the picric acid solution for a second.

8. Dehydrate in absolute alcohol; xylol; balsam.

3. =Unna’s Method for Collagen.=

1. Harden in absolute alcohol; imbed; cut.

2. Stain 5-15 minutes in polychrome methylene-blue.

3. Wash in water.

4. Differentiate in 1 per cent neutral orcein in absolute
alcohol, 15 minutes.

5. Dehydrate in absolute alcohol.

6. Clear in xylol; mount in balsam.

Nuclei dark blue; protoplasm light blue; collagen dark red;
plasma-cell granules greenish-blue; mast-cell granules red.

4. =Mallory’s Fibroglia Method.=

1. Fix thin, small, fresh pieces of tissue in Zenker’s fluid;
harden in alcohol; imbed in celloidin or paraffin; cut.

2. Stain sections in 1 per cent aqueous acid fuchsin for 12 hours
in the cold, or 20-30 minutes at 50-56°C.

3. Wash in water for 5 seconds.

4. Differentiate in 0.25 per cent aqueous potassium permanganate
solution 10-20 seconds.

5. Wash in water for 5 seconds.

6. Dehydrate in alcohol; clear in xylol or origanum; mount in
balsam.

Fibroglia fibrils and cell-nuclei intensely red; contractile
elements of striped muscle, smooth muscle, neuroglia fibres,
cuticular surfaces of epithelial cells and fibrin are also red;
connective-tissue fibres brownish-yellow or colorless; elastic
fibres, unless degenerated, bright yellow.

_b._ =Elastic Fibres.= Weigert’s method is so superior to the Unna
orcein-stain that it alone is given here. It is our best elective
stain: gives permanent preparations, and is in every way practical.
The stain keeps well.

Weigert’s Method for Staining Elastic Fibres.

_Preparation._ Boil in a porcelain dish resorcin 4 grms., fuchsin
(Grübler) 2 grms., and water 200 cc. After the mixture has
boiled a few seconds add 25 cc. of liquor-ferri sesquichlor.,
Pharm. Germ. III. Stir well and boil for 5 minutes. When cool,
filter. Carefully loosen the filter from the funnel, transfer it
to the same porcelain dish which still contains a small amount
of sediment, and add 200 cc. of 94 per cent alcohol. Boil and
stir carefully. Remove the filter-paper when all the sediment is
dissolved. Cool, filter; make up the filtrate to 200 cc. with 94
per cent alcohol, and to these 200 cc. add 8 cc. of hydrochloric
acid. Resorcin-fuchsin may be obtained from Grübler, but the
freshly-prepared stain gives better results.

1. Fix in any ordinary solution; imbed; cut.

2. Stain with lithium-carmine and differentiate in acid alcohol;
wash thoroughly.

3. Stain in the resorcin-fuchsin mixture for 20-60 minutes.

4. Wash rapidly in acid alcohol.

5. Dehydrate and differentiate in absolute alcohol until section
is red.

6. Clear in xylol; balsam.

Nuclei are red; elastic fibres blue-black. Should the stain when
old give a diffuse staining differentiate for a longer time in acid
alcohol.

_c._ =Fat Tissue.= Use same methods as advised for the
demonstration of fatty degeneration and infiltration (osmic acid,
scharlach R, sudan III).

V. EAR.

Remove temporal bone; fix in formol-Müller’s; decalcify in
trichloracetic acid; wash; after-harden in alcohol; imbed in
celloidin. For nerve-endings use Golgi’s and methylene-blue methods.

VI. EYE.

Fix in Müller’s, formol-Müller’s, Zenker’s, formol, Flemming’s
or Marchi’s solution. Aid fixation by incisions into sclera. The
eye should not be left in formol for more than 3 days. Section as
desired; imbed larger pieces in celloidin, small ones in paraffin.
Use alum-carmine, iron-hæmatoxylin, Van Gieson’s, Weigert’s
elastic stain, Levaditi’s silver-method, Golgi’s nerve-stain,
methylene-blue method, etc., according to the object of the
investigation.

VII. LIVER.

For the demonstration of the bile-capillaries _Weigert’s neuroglia
method_ gives the best results. (See Page 300.) This method may be
used with sections cut on the freezing-microtome after fixation
in formol. Such sections are placed in a ½ per cent solution of
chromic acid for 1 hour, transferred to the neuroglia mordant for
5-6 hours, washed well with water, and then treated as for the
neuroglia method. Van Gieson’s method may also be used for frozen
sections of formol-fixed tissue. The walls of the capillaries show
as fuchsin-colored streaks.

VIII. MUSCLE.

Van Gieson’s is the best general stain for both striped and
unstriped muscle, as it differentiates the muscle perfectly from
the connective-tissue. Mallory’s reticulum stain may also be
used for the same purpose. For the study of myoglia fibrils the
tissue must be fixed within a few minutes after its removal from
the living body. Autopsy material cannot be used. These fibrils
can be demonstrated by Mallory’s fibroglia stain, or by Mallory’s
phosphotungstic-acid hæmatoxylin stain for neuroglia. (See below.)

IX. NERVOUS SYSTEM.

It is impossible in a book on general pathologic technic to consider all of the numerous staining methods that have been devised for the study of the nervous system. I have attempted, therefore, to pick out the best selective methods for the staining of the more important nervous structures, so as to cover adequately the general held of nervous pathology. Formol has now replaced Müller’s for the preliminary fixation of nervous tissue, because of its quick action, and because after its use chromic acid may be employed to mordant the tissue, when it is desired to use certain staining methods requiring such mordanting. _Celloidin imbedding_ is preferable, although paraffin may be used for general work. _General stains_, such as _hæmatoxylin and eosin_, and _Van Gieson’s_ are used for general impressions.

1. =METHODS FOR STAINING GANGLION CELLS.=

A. Lenhossék’s Method.

1. Fix in equal parts of saturated watery picric acid and
mercuric chloride (Rabl’s mixture); after-harden in absolute
alcohol; imbed in paraffin; cut.

2. Stain in saturated watery solution of toluidin blue, or
thionin blue, for 12 hours.

3. Wash rapidly in water.

4. Differentiate carefully in absolute alcohol, or in
aniline-alcohol (1-10).

5. Carbol xylol; xylol (quickly); balsam.

Nissl’s granules are blue. This method is easy, and the best for
general work.

B. Nissl’s Method.

1. Fix in 96 per cent alcohol for 2-5 days; mount tissue in
gum arabic on block; harden in 96 per cent alcohol; cut; place
sections in 96 per cent alcohol.

2. Stain in methylene-blue soap mixture (methylene-blue B 3.75
grms., Venetian soap shavings 1.75 grms., water 1,000 cc. Shake
well. Keep 3 months before using. Shake and filter before
using.), warming, until bubbles arise.

3. Differentiate in aniline alcohol (aniline oil 10 parts, 96 per
cent alcohol 90 parts) very rapidly.

4. Arrange section on slide; dry with blotting-paper; cover with
cajuput oil.

5. Blot; wash off oil with benzene.

6. Remove benzene; mount the wet section in xylol colophonium,
slightly warming; press on cover, and remove excess of
colophonium.

Nuclei of ganglion cells light blue: granules dark blue. Toluidin
blue, thionin, dahlia, Bismarck brown or neutral red may be used
instead of methylene-blue, and often give better results.

2. METHODS FOR STAINING MYELIN SHEATHS.

A. Weigert’s Method.

1. Fix in formol 2-3 days.

2. Primary mordant (potassium bichromate 5 grms., fluorchrom 2.5
grms., water 100 cc.: boil and filter) 4-6 days.

3. Without washing after-harden in graded alcohols, in the dark.

4. Imbed in celloidin.

5. Secondary mordant (neutral copper acetate 5 grms., fluorchrom
2.5 grms., water 100 cc., boil and add 36 per cent acetic 5 cc.)
for 1 day at 37°C.

6. Transfer to 70-80 per cent alcohol.

7. Cut.

8. Stain in Weigert’s iron-hæmatoxylin, 24 hours.

9. Wash in water, 30 minutes or longer.

10. Differentiate in borax-potassium ferricyanide (potassium
ferricyanide 2.5 grms., borax 2 grms., water 100 cc.) until the
gray substance appears yellow to white. Control under microscope.

11. Wash thoroughly in water.

12. Dehydrate in absolute alcohol; clear in xylol; mount in
balsam.

Myelinated fibres, blue-black, upon a colorless or light yellow
background; red blood cells may be blue-black. Weigert’s method is
better than any of the numerous modifications.

B. Orr’s Osmic-Acid Method.

1. Place fresh tissue from cerebral cortex or cord, not more than
⅛ inch in thickness, in 1 per cent acetic, 2 cc., and 2 per cent
osmic acid 8 cc., for 48 hours. If mixture is darkened at end of
24 hours, renew.

2. Transfer to 10 per cent formol for 3 days, in order to
complete reduction and hardening.

3. Imbed in celloidin or paraffin; cut.

4. Remove paraffin; alcohol; water; differentiate in ⅛-1/12 per
cent potassium permanganate.

5. Transfer to a 1 per cent oxalic acid solution, until sections
become yellowish-green.

6. Wash; dehydrate; xylol; balsam.

Nerves and fat black: tissue yellowish-green. A very reliable
method.

3. STAINING OF AXIS CYLINDERS.

Stain with Van Gieson’s (red), Mallory’s aniline blue method (red),
lithium carmine (red), or

Williamson’s Modification of Bielschowsky’s Method.

1. Fix in Müller’s; imbed; cut.

2. Place sections in 10 cc. of tap water containing a few drops
of formalin, 5 minutes.

3. Wash in water.

4. Place in the following silver bath 5-10 minutes: 3 drops of
liq. ammoniæ (B.P.) are dropped into a test tube. Add 10 per
cent silver nitrate solution, drop by drop, until a brownish
precipitate is formed. Dissolve the latter by adding ammonia,
drop by drop, until the fluid is quite clear. Add tap water up to
10 cc.

5. Wash thoroughly in water.

6. Transfer to the dilute formol solution until sections become
grayish-black (1-3 minutes).

7. Place in the following solution for a few minutes: To 10 cc.
of water add 2 drops of 1 per cent watery solution of chloride of
gold, a few drops of saturated borax solution, and a few drops of
a 10 per cent solution of potassium carbonate.

8. Transfer to a 10 per cent aqueous solution of sodium
hyposulphite for a few minutes.

9. Wash in water; dehydrate in alcohol; clear in oil of cajuput;
xylol; balsam.

Axis-cylinders, intracellular fibrils and Golgi’s network are
stained.

4. =STAINING OF NEURO-FIBRILLAR STRUCTURES.=

These are stained by Bielschowsky’s method, and by acid fuchsin
after fixation with osmic acid. The special methods (Apathy’s
gold method, Bethe’s molybdic method, the silver methods of
Ramen y Cajal and Robertson) have little practical application
in pathologic work, and are used chiefly in the study of normal
histology.

5. =THE STAINING OF NEUROGLIA.=

A. Weigert’s Method.

1. Fix small pieces of fresh tissue in 10 per cent formol for 24
hours.

2. Mordant. 8 days at room temperature (4 days at 37°C.) in
copper acetate 5 grms., fluorchrom 2.5 grms., acetic acid 5 cc.,
water 100 cc.

Or, harden and mordant at the same time in 9 parts of the copper
mordant, and 1 part of commercial formol for 8 days, changing on
the second day, and once again later.

3. Wash in water: after-harden in alcohol; imbed in celloidin;
cut.

4. Place sections in ⅓ per cent aqueous solution of potassium
permanganate.

5. Wash in two changes of water.

6. Place in the following reducing mixture, 2-4 hours: Chromogen
5 grms., formic acid (sp. gr. 1.2) 5 cc., water 100 cc.; filter;
to 90 cc. add 10 cc. of 10 per cent sodium sulphite solution just
before using.

7. Wash twice in water.

8. Place sections in 5 per cent carefully filtered aqueous
chromogen solution 10-12 hours. (The glia fibres become darker,
and a yellowish contrast is obtained for the ganglion and
ependymal cells and thicker axis cylinders. Connective-tissue is
stained red.)

9. Wash in water.

10. Place section on a slide freshly cleaned with alcohol; dry
with filter paper; stain in the following mixture for about 30
seconds: Saturated solution of methyl violet in 70-80 per cent
alcohol 100 cc., oxalic acid 5 per cent solution, 5 cc.

11. Remove excess of stain; dry with filter paper; cover slide
with saturated solution of iodine in 5 per cent potassium-iodide
solution, 30 seconds.

12. Remove iodine solution; dry with filter paper; differentiate
in a mixture of equal parts aniline oil and xylol until no more
heavy clouds of stain are given off. Control under microscope.

13. Dry section with filter-paper; add xylol; blot; repeat three
times.

14. Mount in balsam or turpentine colophonium.

Neuroglia fibres and nuclei, blue; connective-tissue, blue-violet;
thicker myelin sheaths, ganglion and ependymal cells, yellowish.
This is the best method, none of the modifications giving as good
results. No method, however, will stain every neuroglia-fibre.

B. Mallory’s Neuroglia Method.

1. Fix small pieces in 10 per cent formol, 4 days.

2. After-harden in saturated watery picric solution 4-8 days:
or combine 1 and 2 by using formol 10 cc. with 90 cc. saturated
picric acid solution.

3. Place in a 5 per cent aqueous solution of ammonium bichromate,
4-7 days at 37°C., changing solution 011 second day; or 3-4 weeks
at room-temperature.

4. Without washing, harden in alcohol; imbed in celloidin or
paraffin; cut.

5. Place sections in ¼ per cent aqueous solution of potassium
permanganate, 15-30 minutes.

6. Wash in water.

7. Immerse in 5 per cent aqueous oxalic acid, 5-30 minutes.

8. Wash in several changes of water.

9. Stain in following solution, 1-several days: Hæmatoxylin 0.1
grm., 10 per cent phosphotungstic acid 20 cc., hydrogen peroxide
0.2 cc., water 80 cc.

10. Wash rapidly in water.

11. Differentiate in freshly prepared 30 per cent alcoholic
solution of ferric chloride, 5-20 minutes.

12. Wash in water.

13. Dehydrate in 95 per cent and absolute alcohol or blot with
xylol.

14. Clear in xylol; balsam.

When Zenker’s fixation is used, omit 2 and 3, and after cutting
sections treat with Lugol’s to remove mercury and then with 95 per
cent alcohol to wash out iodine; then wash in water and proceed
with 5.

Neuroglia, nuclei and fibrin, dark-blue; all else is pale yellow or
gray. If the differentiation in 11 is omitted, the axis-cylinders
and ganglion-cells are rose-pink; the connective-tissues, dark
red-pink.

6. =COMBINED STAINING OF SEVERAL NERVOUS STRUCTURES.=

Various methods of impregnation with silver, gold or lead are used
in histologic work, the Golgi methods and their modifications in
particular. They have but little application in pathologic work,
and for that reason are omitted here, as is also a consideration of
Ehrlich’s vital methylene-blue method and its modifications. Full
details of these methods can be found in laboratory textbooks on
histology.

7. =METHODS FOR THE DEMONSTRATION OF NERVE-DEGENERATION.=

A. Marchi’s Method.

1. Harden small fresh pieces of tissue in Müller’s fluid for at
least 8 days. Handle tissue very carefully to prevent mechanical
injury. The tissue may be placed first in formol, and then later
transferred to Müller’s fluid.

2. Place in freshly prepared Marchi’s fluid (Müller’s fluid 2
parts, 1 per cent osmic acid solution 1 part) for about 8 days in
the incubator at 37°C. The brain requires a longer time. When the
mixture loses the osmic acid smell renew it.

3. Wash in running water, 24 hours.

4. Harden in graded alcohols.

5. Imbed in celloidin; cut; dehydrate; clear; mount.

Degenerated nervous tissue (fat) is black: all else brownish gray.
Contrast stain in Van Gieson’s, lithium carmine, etc. This method
is good for the demonstration of early degenerations.

B. Donaggio’s Methods for Early Degeneration.

Method I—

1. Fix in Müller’s fluid or in 4 per cent potassium-bichromate
solution. The tissue may remain in the fluid for any length of
time.

2. Transfer directly, without washing, to alcohol. Dehydrate;
imbed in celloidin; cut sections 20-30 microns. Place sections in
distilled water for a few seconds.

3. Transfer to the following mixture for 10-20 minutes: To 20 per
cent solution of ammoniated chloride of tin add an equal amount
of 1 per cent aqueous hæmatoxylin. Allow to stand for five days.
Keep in the dark, and in a cool place.

4. Wash rapidly in distilled water.

5. Differentiate in Pal’s solution (oxalic acid 0.5 grm.,
potassium sulphite 0.5 grm., water 100 cc.) until the normal
fibres are entirely decolorized.

6. Dehydrate; xylol; neutral balsam.

Degenerated fibres blue; normal, decolorized.

Method II—

1. Fix in Müller’s fluid: imbed as in Method I.

2. Place sections in 0.5-1 per cent aqueous hæmatoxylin solution,
10-20 minutes.

3. Transfer directly to a saturated aqueous solution of neutral
acetate of copper, 30 minutes. Renew copper solution once.

4. Decolorize as in Method I.

5. Wash rapidly in distilled water.

6. Dehydrate in graded alcohols; xylol; balsam.

Degenerated fibres black; normal fibres unstained, except for a
narrow circle at periphery.

Method III—

1. Fix and imbed as in Method I.

2. Stain in 0.5-1 per cent aqueous hæmaloxylin solution, 10-20
minutes.

3. Transfer directly to 10-20 per cent solution of perchloride of
iron. The section becomes black. After a few seconds they lose
their color. If washed in water, they regain their color.

4. Without washing, differentiate in acid alcohol (0.75 cc. HCl
in 100 cc. alcohol).

5. Dehydrate in absolute alcohol; xylol; balsam.

Degenerated fibres appear as small black streaks or circular areas.

C. Staining of Fat-granule Cells.

Fix in Flemming’s or Marchi’s mixtures; or in formol, staining with
sudan III or scharlach R. The tissues may be examined also in the
fresh condition.

D. Old Degenerations.

Use Weigert’s myelin method to show absence of myelinated fibres.
Van Gieson’s method stains the neuroglia of degenerated areas a
deep red; it is very useful combined with Weigert’s myelin stain.
Weigert’s neuroglia stain may be used to demonstrate the relative
parts played by neuroglia and connective-tissue in the formation
of sclerotic patches. When Weigert’s iron-hæmatoxylin is used
with Van Gieson’s the neuroglia remains unstained, while the
connective-tissue stains red. With other hæmatoxylins the neuroglia
cannot be sharply differentiated from connective-tissue when
stained with Van Gieson’s.

8. =PERIPHERAL NERVES.=

Use any of the above methods for the staining of myelin sheaths,
ganglion cells, axis cylinders, etc. Van Gieson’s is good for the
demonstration of connective-tissue increase. For the demonstration
of peripheral fibrils and nerve-endings consult textbooks on
histology for Golgi methods, Ehrlich’s vital methylene-blue method,
and the modifications of May, Drasch, and others.

Platner’s Method.

1. Harden in 25 per cent solution of liq. ferri sesquichlor., 1-5
days.

2. Wash in water, until the addition of KCNS to the water yields
no reaction.

3. Place in 75 per cent alcohol containing an excess of
di-nitroresorcin, 2-30 days, according to the size of the piece
of tissue.

4. Dehydrate in absolute alcohol.

5. Imbed; cut; dehydrate; clear; mount.

Axis cylinders, emerald green. A good method for the rapid
demonstration of pathological processes connected with the
peripheral nerves.

9. =CHROMAFFINIC TISSUES.=

Wiesel’s Method.

1. Fix 1-4 days in 10 parts of a 5 per cent potassium bichromate
solution, 20 parts of 10 per cent formol, 20 parts distilled
water.

2. 1-2 days in 5 per cent potassium bichromate.

3. Wash thoroughly in running water, 24 hours; harden in graded
alcohols; imbed in paraffin.

4. Stain sections in a 1 per cent aqueous toluidin-blue or
water-blue solution.

5. 5 minutes in tap-water.

6. Stain 20 minutes in a 1 per cent watery safranin solution.

7. 96 per cent, and then absolute alcohol, until the blue color
appears.

8. Xylol; balsam.

Chromaffinic cells green; other cells light blue, nuclei red.

X. SKIN.

Skin should be fixed in formol-Müller’s or formol, and should
not be left too long in alcohol or xylol. Imbed in celloidin
preferably. For general use the ordinary stains suffice; for the
study of pigment stain with alum or lithium-carmine. Use Weigert’s
elastic-tissue stain for the demonstration of elastic fibrils.
The intercellular bridges may be demonstrated by Van Gieson’s
(remaining unstained), or by various special staining methods.

Herxheimer’s Method for Epithelial Fibrillae.

1. Harden; imbed; cut.

2. Stain in a saturated watery solution of kresyl-echt-violett.

3. Dehydrate in absolute alcohol; clear in clove oil; balsam.