The Use of Procion Dyes for Light
Microscopy of the Frog Lens
J. L. Rae, K. D. Truirr, and J. R. Kuszak
Protocols for light microscopy of frog lenses that result in good visibility of cell structure from the
lens surface to its nucleus are presented. The lenses are fixed in 10% neutral formalin in 0.06 M
phosphate buffer and are embedded in Epon 812. Following staining of the thick sections with selected
Procion dyes, essentially every cell in the section plane of the lens can be visualized by simple light
microscopy without fluorescence. The methods and dyes allow measurement of cell dimensions at all
depths in the lens and allow investigation of cell packing geometry. The techniques should be generally
useful for studying normal lens structure and the alteration of structure induced by cataractogenesis.
Invest Ophthalmol Vis Sci 24:1167-1171, 1983
easily if large areas of a single lens could be viewed
at one time. We found that the use of formalin fixation and Procion dyes without fluorescence allowed
the morphologic investigation of large areas of lens
surface while minimizing dimensional changes during the fixation process. It is the purpose of this paper
to describe these techniques and to present results
from the frog lens.
Procion dyes: Procion dyes are mono- or dichloros-triazinyl dyes that react with cellulose under mild
conditions in water. Little is written about these dyes
because of their proprietary use in the textile industry.
In the life sciences, they are most known for their
applications in intracellular and extracellular staining. They are useful for this purpose because these
dyes bind to cell structures and remain bound during
preparation of the tissue for microscopy. Also, many
of the dyesfluoresce.What little is known about their
chemistry publicly comes from an article by Stead.3
In general, the dyes are produced by reacting a chromogen molecule with cyanuric chloride as shown in
Figure 1A. The resulting compound, because of the
greater negativity of the chlorine in comparison with
carbon, has polarized carbon-chlorine bonds that
make it subject to nucleophilic attack. Reactions with
amino groups of proteins occur readily in water solutions of pH 10-11 and apparently to a lesser but
appreciable degree at pH 7.4. The schematic reaction
is shown in Figure IB. This reaction has made these
dyes useful as staining agents for protein electrophoresis.* The dyes are provided as either an H series or
an MX series, this classification depending on the
presence of monochloro or dichloro groups respec-
Light and transmission electron microscopy (TEM)
of the nonembryonic crystalline lens are extremely
difficult. Although little has been published to allow
one to document this difficulty, the problems involved in doing lens morphology are well known
among workers in the field. Most investigators who
present light microscopy or TEM of the lens use one
of two approaches. First, some work with small pieces
of tissue dissected from either a fresh lens or a partially fixed lens.1 This technique, although it ensures
fixation and plastic infiltration into the tissue, is troublesome because it produces an unknown degree of
dissection damage and it requires the processing of
many such pieces of tissue in order to sample adequately the structure of the tissue. Second, some use
the entire lens but only present micrographs from the
superficial layers where fixation and plastic infiltration are reasonably assured.2 This technique circumvents the problem of tissue dissection damage but of
course does not allow study of structures deep in the
lens.
Techniques allowing visualization of morphology
from large areas of a single lens would be useful since
naturally occurring cataracts and most induced cataract models result in regions of opacity that involve
an appreciable fraction of the total lens volume. In
addition, an understanding of the organization and
structure of the normal lens would be obtained more
From the Department of Physiology, Rush Medical College,
Chicago, Illinois.
Supported by grants from the NIH EY03282, The Louise C.
Norton Trust, and the Regenstein Foundation.
Submitted for publication: June 1, 1982.
Reprint requests: J. L. Rae, Department of Physiology, Rush
Medical College, 1750 W. Harrison, Chicago, IL 60612.
* Polysciences Inc., Warrington, PA. Data sheet #182.
0146-0404/83/0900/1167/$ 1.05 © Association for Research in Vision and Ophthalmology
1167
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INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / Seprember 1983
Cl
Cl
Dye-NH2-f-
Dye"
NH-Protein
1
Dye
ci
c
NH-Protein
Fig. 1. A, Schematic reaction for the formation of a Procion dye
from a chromogen and cyanuric chloride. B, Schematic reaction
of Procion dyes with protein amino groups.
tively. We found selected dyes from both series to be
useful and expect that these dyes have general application as histologic stains.
Materials and Methods
All experiments were done on small Rana pipiens
whose lens equatorial diameters varied between 3 and
4 mm. The lenses were prepared by removing the eye
from the animal and excising the posterior part of the
globe under Ringer solution, leaving the lens in the
eye suspended from its zonules. This preparation was
immersed in thefixativeat room temperature for one
hour to allow the zonules and outer lens material to
fix. The lens was subsequently removed from the
globe with a lens loop and was reimmersed in the
fixative solution.
The fixative was 10% neutral formalin (actually
3.8% formaldehyde) with 1% tannic acid in 0.06 M
sodium phosphate buffer at pH 7.3. Thisfixativepreserves lens dimensions during the fixation process
(Rae and Stacey, unpublished observations). The
lenses were fixed for 72 hrs at room temperature,
washed for 24 hrs in 0.06 M sodium phosphate buffer
and then dehydrated in ethanol. The preliminary dehydration protocol was 15 min each in 30%, 50%,
70%, and 95% ethanol. Next, they were subjected to
three 15-min changes of 100% ethanol and left overnight (12 hrs). They were subsequently immersed for
15 min each in 3:1, 1:1, and 1:3 mixtures of
ethanol:propylene oxide followed by 100% propylene
oxide for 1 hr. They were next placed in 3:1 propylene
oxide:Epon 812 for 1 hr and were left overnight in
1:1 propylene oxide:Epon 812. The next day they
were placed in a 1:3 mixture of propylene oxide:Epon
812 for the entire day (8 hrs) after which they were
placed in pure Epon 812 and left overnight. The
lenses were embedded in pure Epon in either flat
embedding molds or in gelatin capsules and were
polymerized in a 70° centigrade oven for 48 hrs. The
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Vol. 2 4
Epon mixture used 13.1 g Epon 812, 4.0 g DDSA,
13.0 g NMA, 0.43 ml BDMA. This extremely lengthy
and tedious protocol was necessary to ensure nuclear
fixation, dehydration, and plastic infiltration.
The lenses were sectioned on a Sorval MT-2B ultramicrotome using glass knives. The 1-2 /*m thick
sections were stained according to the following protocol:
The section was placed on a drop of water on a
coverslip and heated on a 90 C hotplate for 5 min.
After cooling, one drop of the Procion mixture was
placed on the section that was then reheated for about
5 sec or until the first sign of a dry ring of stain surrounding the tissue. The section was washed with
deionized water and blown dry with a clean air
source. One drop of the counter stain was placed on
the section, which was then heated for 20 sec. Following washing and drying, as above, the coverslip
was mounted to the slide with Harleco Krystalon.
The Procion stain mixture was 1 part of a 6%
aqueous solution of Procion orange MX-G with 1%
sodium borate andfiveparts of a 6% aqueous solution
of Procion red MX-8B with 1% sodium borate. The
counterstain is a 1:1 mixture of aqueous solutions of
1% methylene blue with 1% sodium borate and 1%
azure II with 1% sodium borate.
Photographs were obtained through a Nikon Labophot microscope with planapochromatic objectives
and a Tiffen #12 yellow filter.
Results
Fixation of the frog lens for light microscopy can
be achieved adequately with a formaldehyde-based
fixative. Following an overnight fixation in 10% neutral formalin in 0.06 M phosphate buffer, individual
lensfiberscan be teased from the center of a 3-4 mm
equatorial diameter lens and thus they appear stabilized
in structure. A fixation time of 72 hrs was chosen,
however, since overnight fixation did not allow the
complete dehydration or plastic infiltration achieved
with the 72-hr protocol. Other fixation protocols such
as 72 hrs in 2.5% glutaraldehyde in 0.05 M sodium
cacodylate or 12 hrs in an 8:2 mixture of 2.5% glutaraldehyde in 0.05 sodium cacodylate:3% acrolein in
0.05 M sodium cacodylate are about as effective by
this microdissection criterion, but such lenses are much
more difficult to stain with the Procion dyes. Therefore,
we chose to use the formaldehyde protocol for the
studies reported here. The nucleus of properly fixed
lenses did not artifactually separate from cortex during
dehydration, a separation that always occurred when
the center was not fixed. It was impossible to achieve
plastic infiltration into the center of lenses where this
artifactual separation of nucleus and cortex had occurred during dehydration.
No.
PROCION DYES AND LEN5 MICROSCOPY / Roe er ol.
Fig. 2. Light photomicrographs
from a montage of a Procion
stained, 1 /*m thick section from
frog lens equator. Every third micrograph of the montage is depicted. Arrows point to cell specializations often associated with
cell column convergence.
We tried a number of plastics including JB-4,
Epon-Araldite, Spurr, Ladd-Ultra Low Viscosity, and
water soluble Durcopan. Although infiltration to the
lens center could often be obtained with any of these
plastics and could usually be obtained with JB-4,4 the
combination of infiltration and stainability of sections was routinely best with Epon 812. For lenses
of the size used for this study, whole lens sections
which passed through the lens center were possible
along any plane including the equatorial plane which
contains the largest amount of tissue.
Using the procedures that we have described, one
can routinely construct montages extending from the
frog lens surface to its center (Fig. 2). In this figure,
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composed of every third micrograph of the montage,
every cell in the cross section can be visualized clearly.
The cells in the surface zone stained much better
and were more clearly outlined when the Procion
staining step was omitted and only the Toluidine
blue-azure II-Sodium Borate counterstain was used.
However, this counterstain so heavily stained the inner zone that no structure could be discerned there.
The Procion stain seemed to keep this counterstain
from being so intense and thus allowed visualization
of the deep structures.
Using montages or using the slide directly under
the microscope, thicknesses and widths can be measured from cells at every depth in the lens. Figure 3
INVESTIGATIVE OPHTHALMOLOGY 6 VISUAL SCIENCE / September 1983
1170
400
600
Depth
Fig. 3. Plot of fiber thickness (•) and fiber width (•) as a function
of depth for fibers in the equatorial plane of a typical frog lens of
1.5 mm radius. Measurements are given to a depth of 1200/mi only
because of the possibility of dimensional distortion at deeper locations
caused by a section not going perfectly through the lens center.
shows fiber width and thickness measurements from
a typical lens plotted as a function of depth in the
lens.
The apparent columnar structure of thefiberlayers
is quite imperfect. In order to have a perfect columnar
structure, the lens would have to contain the same
total number of cells in each concentric spherical
shell. Since the circumference of each shell increases
with its radial position from the lens center, cells in
each more superficial layer would have to be greater
in average width in order for the total number of cells
to remain the same. Although it is clear from Figure
3 that cells in more superficial layers are slightly wider
on the average than cells deep in the lens, it is also
clear that the number of cells in more superficial layers must increase since the circumference of superficial shells (calculated as 2irr where r = distance from
lens center) increases faster than does the cell width.
The lens appears to maintain its quasi-columnar
structure by occasionally producing larger cells that
bridge across two to three more superficial columns.
This structure is clearly depicted in Figure 2 (arrows).
These structures are seen at all depths in the lens and
attempts are currently underway to map their occurrence precisely.
Figure 4 is a montage that clearly depicts the radial
structure of the frog lens near its center. In this region,
Fig. 4. Montage from the center of a 1 jum thick equatorial section of frog lens.
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Vol. 24
No. 9
PROCION DYES AND LENS MICROSCOPY / Roe er ol.
the lens fibers are in almost perfect concentric shells
with each adjacent fiber offset from its neighbors by
Vi a fiber thickness. This concentric shell orientation
is maintained throughout most of the lens, but the
curvature of the more superficial layers is not so obvious because of the increasing radius of curvature
of layers progressively nearer the surface.
Discussion
This method for light microscopy of lenses, with
a modification of the buffer osmolarity, also works
well for rat, mouse, chick, and fish lenses and, thus,
probably has general usefulness for lens morphology.
The procedure allows preservation of the entire lens
for lenses with equatorial diameters of 4 mm or less.
We have not yet tried the technique for larger lenses.
The methods presented are sufficiently valuable
that they may be of considerable use in studies of
experimental cataracts and normal lens morphology.
These studies suggest that at least in small lenses,
differential staining and not fixation may be the pri-
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mary problem in trying to do quality light microscopy
of large areas of lens tissue. We have no reason to
believe that Procion dyes are unique in their ability
to stain lenses differentially. Additional experimental
effort to identify other and perhaps more effective
staining procedures would seem important to pursue.
Key words: crystalline lens, morphology, Procion dyes, light
microscopy, neutral formlin, plastic embedding, frog
References
1. Kuwabara T, Kinoshita JH, and Cogan DG: Electron microscopic study of galactose-induced cataract. Invest Ophthalmol
8:133, 1969.
2. Rae JL and Stacey TR: Intracellular markers in the crystalline
lens of the rat. Doc Ophthalmol Proc Ser 8:233, 1976.
3. Stead CV: The chemistry of reactive dyes and its relevance to
intracellular staining techniques. In Intracellular Staining in
Neurobiology, Kater SB and Nicholson C, editors. New York,
Springer-Verlag, 1973, pp. 41-59.
4. Datiles M, Fukui H, and Kinoshita JH: Galactose cataract
prevention with sorbinil, an aldose reductase inhibitor: a light
microscopic study. Invest Ophthalmol Vis Sci 22:174, 1982.