THE APPLICATION OF THE FREEZIXG-DRYING TECHNIQUE
         TO RETINAL HISTOCHENISTRY

CHRISTIAN B. ANFINSEX, OLIVER H. LOWRY AND A. BAIRD HASTINGS
Department of Biological Che?uistry, Harvard 3fedicaZ School, Boston, Massachusetts

THREE FIGURES

I?;TRODUCTION

In a quantitative histochemical study of retina, it is essential that
serial sections containing predominantly one retinal layer be obtained.
Due to the fact that the structure of the retina changes radically every
40-50 micra, the method of Linderstrdm-Lang and blogensen (`38)
which employs alternate sections for chemistry and histological control
cannot be used. The slight crumbling which occurs during the sectioning
of this frozen tissue also prohibits the use of this technique since a
fundamental requirement of the Linderstrtim-Lang and Mogensen
method is that all sections be of uniform area and weight. A procedure
has consequently been evolved which embodies the main principles of
this method and those of the Altman-Gersh freezing-drying technique
(Gersh, `32), permitting the use of the same section of tissue both
for identification of cell type present and for chemical determinations of
certain tissue constituents. The method offers advantages over present
freezing-dryin g techniques in that the process of dehydration can be
carried out in a much shorter time and with simpler equipment and that
paraffin embedding is avoided. The principle consists essentially of the
following steps: the rapidly frozen tissue is mounted directly on the
disc of an object holder; frozen sections are cut on the microtome in
the constant temperature freezing chamber of Linderstrdm-Lang and
llogensen, and are dehydrated at atmospheric pressure over phosphorus
pentoxide in the same chamber. Each section is then stained with a
~~101 solution of methyl violet, and finally, after histological observation
has been made, the section is employed for the chemical determination.

Emyme activity of stnilzed frozemdried sectioas. Experiments were
carried out to test the effect of the procedure on the stability of various
enzyme systems. As examples, peptidase activity and diphosphopyridine
nucleotide assay of rat liver and choline esterase of rat brain cortex
have been studied.

231


232      C. B. AKFIKSES, 0. H. LOWRY ASD A. B. HASTIXGS

Peptidase was determined by the micro method of Lilldcrstrlrnl-l,~~ll~
and Halter ( `35), diphosphopTridine nucleotide by a modification of tllcl
method of Jandorf, Klcmpcrer and Hastings ( `Al), employing the
Cartesian diver technique of Linderstrdm-Lang ( `3i), and clloliiie
&erase by the method of Glick (`38). In the case of peptidasc, slight
reduction in activity was noted, the greatest decrease in activity amount-
ing to less than 10% when the determinations were carried out within
24 hours after dehvdration. In t,he case of the other substances tested no
diminution in activity was observed. Data showing the agreement bc-
tween the choline esterase activity of frozen-dried, stained sections of'
rat brain cortex and fresh frozen tissue are presented in figure 1.

X - FROZEN-DRIED, STAINED SECTIONS

30           60           90
     MINUTES

Fig. 1 Choline esternse activity of frozen-dried, methyl riolet stained sections, and fresh
frozen section8 of rat brain cortex.

The incubation mixture in each vessel consisted of one OO-micra, circular section of rat
hraiu cortex, 3 mm. in diameter, 10.6 mm". of 307; glycerine, and 13.1 mm". of substrate
(Glick, `38).

It is observed that the dryin, 0 and staining procedure involved has
no effect on the choline esterase activity of the tissue. Alternate sections
of the rapidly frozen liver or brain cortex were employed as controls
in the case of peptidase and choline esterase respectively. In the CRW
of diphosphopyridine nucleotide, the micro assays were compared with
values obtained in this laboratory by the method of Jandorf et, al.,
employing the Warburg manometric technique. The rate of CO2 evolu-
tion per microgram of DPN as det,ermined by the micro- and macro-
methods was found to be identical.

GENERAL TECIISIQVE

Freezing. It has been shown by Hoerr (`36) that rapid freezing is
essential in the prevention of ice crystal growth in the preparation of
tissue for histological work. To prevent distortion from this cause.


HISTOCHEMICAL TECHNIQCE FOR RETISA         233

s;lnall samples of tissue must be employed and rapid stirring of the
freezing mixture is necessary. For accurate histological control, there-
fore, the following procedure has been usecl. As soon as possible after
the death of the animal, a small piece of tissue is removed and dropped
illto isopentane previously chilled to a semi-liquid state by immersion
ill liquid nitrogen. The isopentane is immediately stirred vigorously.
Xozclltillg. The tissue is then remo\:ecl, trimmed to a cube roughly

;ll)out 0.3 cm. on a side, and placed on a small object holder which
ilas been moistened with a film of blood. (An ordinary flat-headed bolt
ulap be used for this purpose.) The whole block is immediately im-
mersed in a mixture of carbon dioxicle snow (dry ice) and petroleum
ether until its transfer to the microtome thermostat. An alternative
procedure consists of placing the tissue block on the object holder,
allowing the portion adjacent to the warm metal to thaw, and, before
further thawing can take place, immersing the entire block in the
Freezing mixture at once.

Cliffirlg ad Jehydrtrf iou. Cutting is carried out as described by
Linderstrtim-Lang and Mogensen ( `38). The sections are then trans-
ferred to small desiccators where they are arranged serially on a clean
slicle. With phosphorus pentoxide as the desiccant, 20 micra sections
are completely dehydrated lvitllin 1 to 13 hours at -20oC. For more
rapid dehydration, the sections are dried in vacua over phosphorus
pentoxide. The tissue sections are then quite dry within 15 to 30 minutes.
Since fine histological detail has not been essential in our present
applications of the method, extended experiments on vacuum dehpdra-
tion hare not been carried out.

After dehylration is complete, the desiccators are stored in the
refrigerator until needecl for chemical or histological examination.

StcriGg. To prevent the solution or displacement of the proteins and
of other more soluble constituents, such as electrolytes, coenzymes, and
small organic molecules, and at the same time to prevent the denatura-
tion of enzymes from occurring, it is essential to USC a non-aq~~cous
solvent for staining which does not injure the enzymes to be studied.
It has been found that xylol satisfies the above requirements. To stain
the tissue, one part of a 40-nig. per cent solution of methyl violet in
absolute alcohol to 50 parts of xylol arc usecl. TTllder these conditions,
the cytoplasm is stained violet leavin g the nuclei completely unstaineh
and appearing as clear vacuoles. When a sufficiently deep stain has been
obtained (generally after 10 millutes), the section is washed with xylol
to insure complete removal of the soluble lipoid material, transferred
to a slide, flattened with a cover slip, ant1 a pliotoprapli or camera lucida


234      C. B. ASFISSEK, 0. H. LOWItT AND A. B. HASTISGS

drawing is made, A drop of sylol is then touched to the edge of the
cover slip to diminish its adhesion to the slide, the slip is lifted off, and
the excess xylol removed with filter paper. After drying in air, the
section is ready for chemical or enzyme determinations. It is essential
to remove the sylol-soluble lipids completely, since, otherwise, the
section may adhere strongly to the slide.

Special technique fo,r retina. The eye 1 is removed from the animal as
soon as possible after death (S-10 minutes in our experiments), and
trimmed free of adhering muscle and fat. It is then plunged, optic
nerve down, into the freezing mixture consisting of dry ice and petroleum
ether. This freezing medium has been found to be satisfactory for
our present purposes since the greater histological detail obtained
by more rapid freezing is not needed here (fig. 2).

The posterior one-third of the eye is separated for study with a
chilled saw. With bone-cutting forceps, the frozen vitreous humor is
removed with a few sharp cuts leavin, "3 the retina, attached to choroid
and sclera, intact. The material is occasionally dipped into the freezing
mixture to prevent thawing and to maintain a brittle condition. With the
same forceps and a scalpel, the retina and adherent choroid can now be
chipped off and immediately placed in t.he chilled petroleum ether.
This can be done quite easily because of the natural cleavage plauc
existing between the choroid and sclera.

Well mashed liver which has been frozen to the object holder and
slightly thawed at the surface by the application of a warm spatu'a
is used as a base on which to mount the retina. h small piece of retina
and its adhering layer of choroid is then removed from the freezirlx
mixture, blown on gently to remove the film of petroleum ether, autl
rapidly placed, with the retinal side down, on the liver. To ensure
subsequent adhesion of the retina-choroid sample to the liver durillx
sectioning, the tissue is gently pressed against the liver base with the
chilled blade of a small scalpel. The above operations must be carried
out with considerable rapidity in order to preclude any thawing of the
retina. The block is then immersed at once in the dry ice mixture, alltl
finally transferred to the thermostat. The object holder is mounted 011
the microtome after equilibrating for 1 hour at the temperature of tlJc
thermostat.

In order to obtain sections containing primarily one retinal la:--c~..
the surface of the retina-choroid sample must be oriented as lleaJ'1?.
parallel as possible to the cutting plane. This orientation can be greatl!
facilitated by the use of a small hand-mirror, held above or at the siilc

`Ox eyes have been used exclusively in our experiments.


HISTOCHEMICAL TECHNIQUE FOR. RETINA         235

of the tissue block during the manipulation of the adjusting screws in
the microtome head.

Each section is removed as it is cut because of the great fragility of
the retinal tissue. In doing this, one sacrifices some of the uniformity
of thickness emphasized by Linderstrtim-Lang and Mogensen, but this
uniformity is unnecessary in the present technique because each section
is weighed and studied individually. The analytical results have been
based on the dry weights or lipid-free weights as determined on a
sensitive torsion balance similar in principle to the one described by
Barrett et al. ( `38), except that. torsion is supplied by a quartz fiber
suspension rather than by a tungsten wire, and the displacements are
measured with a cathetometer.

After weighing and staining as described above, the sections are used
directly for chemical study. In some cases, portions of the sections
coutaining oue retiual layer may be dissected out with a small scalpel
under low power magnification.  Such dissection is carried out uudei
sylol.
The method is at present being a pplied to the distribution of choline
esterase in retina. Reproducible correlations are found from experi-
ment to experiment indicating the reliability of the histological control.

APPRAISAL OF THE SECTIOSS OBTAISED

In order to demonstrate that the stainin g teclmiquc employed here
permits adequate histological differentiation for tbc present purposes,
the following comparison with hematoxylin staining was made. Figure 2
shows the results obtained by (a) staining a frozen-dried section of
retina with sylol-methyl violet, and (b) by staining another section in
the series fised in 95% alcohol with hematoxylin. The section on the
left illustrates the fact that methyl violet, dissolved in xylol, has the
property of staining the cytoplasmic elements in the various layers
of the retiua. The stain does not, however, differentiate between oue
cytoplasmic layer and another except in the case of the layer of rods
and cones which stains more deeply than the other zones and, in addition,
contaius pigment granules, particularly in light-adapted eyes. How-
ever, in the course of consecutive examination of a complete series of
horizontal retinal sections, the alteration of stained and unstained layers
is sufficient for their ideutification and correlation with enzyme content.

Figure 3a shows the appearance of rat liver carried through the
freezing-dryiug procedure, unstained, but cleared with sylol. This
photograph illustrates tliat the procedul~e employed iu this study does


2%     C. B. ASFISSES, 0. II. LOWRY ASD A. B. HASTISOS

iiot materially obscure the iioxmal liistolopical picture. It will be ob-
served that the hepatic cells and their nuclei are v-e11 defimd and that
undistorted crytlwocytes appear in typical bepatic sinusoids.

                    a                          b

Fig. 2 Camera lucida drawing of the layers of the retina of an ox as stained by (a) xylol-
methyl violet and (b) hematoxylin; 1, choroid; 2, pigment granules; 3, rods and cones; 4, outer
nuclear lapcr; 5, outer plexiform layer; 6, inner nuclear layer; T, inner plexiform layer. X 120

                 a
Fig. 3  (a) Frozen-dried, xylol cleared rat 1iT
rat liver.

b

rer. (h) Frozen-dried, xylol-methyl violet stnincd


HISTOCHEMICAL TECHNIQUE FOR RETINA         237

Figure 3b is a frozen-dried section of a rat's liver treated with xylol-
methyl violet, t,o illustrate that the cytoplasm of the hepatic cords is
deeply stained, whereas the hepatic nuclei are unstained.

The authors wish to thank Dr. Philip L. Pillsbury for his valuable
assistance in many of the earlier experiments.

A method is presented which permits histological examination and
quantitative enzymatic study of individual frozen-dried sections of
tissue. StaiIjng with methyl violet is carried out in sylol to prevent
the solution and displacement of non-lipoid tissue constituents. The
manipulations involved have a very slight or 110 effect on the enzyme
systems tested. Bp this method, it has been possible to study quantita-
tively enzymatic localization in the various layers of the retina.

LITERATURE CITED

BARRETT, II. JI., C. H. BEST AND J. H. RIDOI-T  1938  A study of the source of liver fat using
     deuterium as an indicator. J. Plrgsiol., y-01. 93, pp. 367-381.
GERSH, I. 1932  The Altmnn technique for fixation by drying while freezing. Anat. Rec.,
     vol. 53, pp. 309-337.
GLICK, D.  1938 A micro method for the determination of choline e&erase and the activit:-
     pH relationship of the enzyme. Compt. Rend. Lab. Carlsberg, vol. 21, pp. 263-268.
HOERR, S. L.  1936 Cytological studies by the Altmnn-Gersh freezing-drying method. I.
     Recent adwnres in the technique. Anat. Rec., vol. 65, pp. 293~31i.
.JAXDORF, B. J., F. W. KLEYPERER ASD A. B. HASTISGS  1941 A manometrir method for the
     determination of dipl~onpl~ol~yridine nucleotide. a. Biol. Chem., l-01. 138, pp. 311-320.
LINDERSTR~M-LUG, K.  1937  Principle of the Cartesian direr applied to gasometric technique.
     Nature, vol. 140, p. 108.
L~NDERSTR@JI-LASG, K., AND IT. HOLTER 1933 The distribution of pcptidase in the gastric
     and duodenal mueosa of the pig. Compt. Rend. Lab. Carlsberg, vol. `70, pp. 4%~.
LINDERSTR~Y-LASG, K ., AND I<. R. MOGESSEX 1938 Histological control of l~istochemical
     investigations. Coml)t. Rend. Lab. Cnrlsberg, vol. 23, pp. 07-34.