T H E
G O M O R I FOR
MARGUERITE A . CONSTANT, P H . D . With the technical assistance of M . Jean Sheets Saint Louis, Missouri
During the course of studying the staining characteristics o f the lens and the lesions o f steroid and diabetic nephropathy, it was o b served that the Gomori aldehyde-fuchsin procedure offered excellent delineation o f many membranes of the eye. T h e staining o f elastic fibers and especially the staining o f secretory granules o f the cells o f the islets of Langerhans by this procedure is well known. However, this stain has received little attention in eye pathology. T h e simplic ity o f the procedure and other characteristics of the stain may warrant its more general use for study of ocular tissues. The present preliminary report notes some observations of interest. METHODS Calottes of eyes, fixed in 10-percent maldehyde were dehydrated, cleared, bedded in paraffin in the usual manner sectioned at 4.0 μ. T h e aldehyde-fuchsin
for em and pro-
* From the Department of Ophthalmology and the Oscar Johnson Institute, Washington University School of Medicine. This research was supported in part by a grant-in-aid from the National Society for the Prevention of Blindness and by grant N B 1375 from the National Institute of Neurological Diseases and Blindness of the National Institutes of Health, U.S. Public Health Service.
cedure used was as outlined in the A F I P Manual,^* which is identical to the original preparation- except that in our studies the Gomori trichrome stain^'' was used as the counterstain. Thus the stains employed were essentially aldehyde-fuchsin, chromotrope 2 R and light green S F . T h e stain works well after Bouin's fixative but tissues may show a pale lilac background after mercury-con taining fixatives or after many months in formaldehyde. Chromate fixatives are said to cause murky shades.^ O u r studies have shown that a 10 to 15-min. dip in 0.1 N H C l , after rehydration and before staining, fol lowed by brief rinsings in water, accentuates the green to blue-green shades o f light green S F dye uptake by connective tissue without impairing the staining o f aldehyde-fuchsin positive fibers. T h e periodic acid leucofuchsin ( P A S ) stain used was that o f Lillie^ with the colloidal-iron method o f Rinehart and Abul-Haj* modified to increase the spec ificity o f the colloidal iron by a 1:4 dilution.= RESULTS Epithelial basement membrane of ciliary body. T h e thickened basement membrane of diabetics" is not homogeneous as indicated
Figs. 1-8 (Constant). The use of Gomori aldehyde-fuchsin stain for ocular tissues. (The photomicrographs were taken from microscopic sections of eyes of four diabetics.) Fig. t. PAS stain of thickened basement mennbrane of epithelium of ciliary body of human diabetic. Fig. 2. Serial section stained with aldehyde-fuchsin which shows intense staining of a narrow portion nearest the epithelial cells with the larger proportion of the thickened membrane being almost unstained. Fig. 3. Drusen of Bruch's membrane stained with PAS which shows the general homogeneity oí staining of the membrane and the drusen. Fig. 4. Drusen of Bruch's membrane stained with aldehyde-fuchsin which shows the continuous inner cuticular lamina cover ing the drusen and the tendency of the drusen to take up other dyes used in the staining procedure. Fig. 5. Descemet's membrane near the trabecular meshwork. Fig. 6. Equatorial region of the lens. Observe the adherent sheet of zonules. The small dark particles are stained debris caught on the surface of the section. Fig. 7. A tangential section which shows zonules attached to the ciliary epithelium end inner limiting membrane. Fig. 8. Optic nerve.
USF. ΟΙ- G D M O R I .AI.DEFIYDF.-KUCHSIN S T . M N
USE OF GOMORI A L D E H Y D E - F U C H S I N STAIN
b y the P A S stain (fig. 1) but consists o f a somewhat thickened, intensely staining por tion o f the membrane adjacent to the pig ment epithelial cells with the major portion of the thickened membrane showing very little dye uptake (fig. 2 ) .
Bruch's membrane and drusen. T h e P A S stain indicates a homogeneity o f the ele ments of Bruch's membrane and the P A S positive drusen (fig. 3 ) . The aldehyde-fuchsin stain shows well the inner cuticular layer and outer elastic layer with pale-lilac o r pale-green staining in between, especially noted in areas o f pathologically thickened membrane or at the ora serrata. T h e con tinuity o f the cuticular layer over a drusen is clearly seen (fig. 4 ) . T h e drusen show varied affinities f o r the dyes much as after staining with the Masson-Goldner trichrome stain.
and the lens cap
sule. These membranes are only faintly stained by aldehyde-fuchsin (figs. 5 and 6 ) . A thin concentration o f aldehyde-fuchsin stain is seen subendothelially in the cornea. T h e zonules stain intensely (figs. 6 and 7 ) . Other areas. T h e internal limiting mem brane of the vitreous is aldehyde positive, as are, o f course, the inner and outer elastic lamellae o f vessels. T h e connective tissue elements o f the optic nerve are nicely differ entiated into aldehyde-positive fibers and fibers which take up the light green S F dye (fig. 8 ) . The junction o f the sclera to the cornea frequently shows a change in stain ing characteristics o f the green sclera to a green-blue admixed with diffuse purple o f the corneal stroma. T h e pigment granules o f the retinal pigment epithelium especially, and those o f the iris stroma occasionally, tend to adsorb the aldehyde-fuchsin, where as the pigment granules o f the choroid, ciliary body pigment epithelium and iris pig ment epithelium generally do not adsorb the dye. T h e procedure results in light bleaching o f pigment which appears light yellowbrown in color. T h e adsorption o f stain b y pigment grantiles may vary somewhat with
the length o f time postmortem before fixa tion, as does the staining o f nuclei and o f the corneal stroma. DISCUSSION T h e aldehyde-fuchsin stain was accident ally discovered by Gomori several years ago to be an excellent stain f o r elastic fibers and ß-cell granules o f the islets o f Langerhans. It has been used primarily f o r staining such structures. In addition, it stains the granules of the gastric chief cells, o f mast cells and some mucins. T h e mechanism o f the stain ing action o f the dye is not known. Although many argyrophilic fibers are also stained by aldehyde-fuchsin, this stain is reported not to stain reticulin." T h e accumulation o f fibers in an arteriosclerotic artery of kidney stains with the same intensity as the inner and outer elastic lamellae. H o w e v e r , a differ ence in these fibers is readily discernible by use of other stains, such as crystal violet, which only stains the elastic lamellae and fibrin, or by examination in ultraviolet light in which case only the elastic fibers show autofluorescence. Similarly the use o f the aldehyde-fuchsin stain has shown a differ ence in the thickened basement membrane o f the epithelium o f the ciliary body from that o f the nephron tubules o r B o w m a n ' s capsule. In these instances the thickened membranes show similarity in their P A S positivity. A more detailed description of these differ ences as shown b y several stains will be presented in a separate report. This observa tion raises interesting speculations as to the cause o f the thickening o f these membranes and suggests that different chemical com positions should be found. T h e aldehyde-fuchsin stain does not offer advantages over the P A S stain or the silver stains except by showing differences in some pathologic structures in the former instance and being more easily and reproducibly used than the latter stains. T h e pastel shades o f the trichrome stain offer subtle contrasts f o r observations on connective tissue which have an advantage over the general pink back-
M A R G U E R I T E Α. C O N S T A N T
ground color of the P A S stain but do not take the place of careful examination of morphology. T h e poor staining o f nuclei is a disadvantage except when one is inter ested primarily in the aldehyde-fuchsin posi tive fibers or membranes. It is felt that the aldehyde-fuchsin stain may be a useful tool for studies of ocular pathology. SUMMARY A
preliminary report on the use o f the
aldehyde-fuchsin stain of Gomori f o r study of membranes of the eye is given. This stain combines the advantages o f staining struc tures stained by P A S o r silver techniques in a simple, reproducible procedure. O f special interest is the observation that the markedly thickened basement membrane o f the epi thelium of the ciliary body of diabetics is not homogeneous as indicated b y P A S stains. 640 South
REFERENCES 1. Armed Forces Institute of Pathology, Manual of Histologic and Special Staining Technics, Wash ington, D.C. (a) p. 82; (b) p. 70. 2. Gomori, G.: Aldehyde-fuchsin: A new stain for elastic tissue. Am. J . Clin. Path., 20:665, 1950. 3. Lillie, R. D . : Histopathologic Technic and Practical Histochemistry. New York, Blakiston, 1954. 4. Rinehart, J. F., and Abul-Haj, S. K . : Improved method for histologic demonstration of acid mucopolysaccharides in tissues. A M A Arch. Path., 52 :189, 1951. 5. Fullmer, H . M . : Effect of peracetic acid on the enzymatic digestion of various mucopolysaccharides: Reversal of the P A S staining of mucin. J. Histol. & Cytochemistry, 8:113, 1960. 6. Yamashita, T., and Becker, B.: The basement membrane in the human diabetic eye. Diabetes, 1 0 : 167, 1961. 7. Pearse, A. G. E . : Histochemistry, Theoretical and Applied. Boston, Little, Brown, 1960, p. 162.
A N D T H E FUNDUS
RoxALD M . BURNSIDE, M . D . , AND CHARLOTTE LANGLEY Dallas, Texas
The measurement o f the percentage o f anisometropia, optical aniseikonia, by the Carl Zeiss fundus camera has been under taken in this study. In the past, the fundus has been measured by viewing it with the ophthalmoscope and using various superim posed grids in the ophthalmoscope to meas ure the blood vessels and the lesions o f the eye. B y ineasuring anisometropia with the fundus camera it is possible to obtain a purely subjective measurement o f the two fundi to compare with the eikonometric readings and the calculated mathematical difference in the refraction. Subjective measurements by the space eikonoineter are not particularly reliable in the very young patient and sometimes in the not so young. In cases o f squint with o r
without suppression, they may be o f no value whatsoever. Thus, the photographs of the fundus that can be measured objectively on a projected screen-grid combination would seeiT! to be o f value. T h e new model Zeiss fundus camera is reflex free and it is possible in patients under 40 years o f age with little or no inacular disturbance actually to visualize the foveal reflex in the finished projected fundus photograph. This fundus camera with its telecentric lens takes very accurate fundus photographs. One hundred normal photographs were made to establish controls f o r this study. Corresponding fundus slides o f the patients, matched f o r size to the right and left eyes, were projected with the Bausch and Lomb