Journal of Neuroscience Methods, 33 (1990) 11-21
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Elsevier NSM 01081
A neurofibrillar staining method for retina and skin: a simple modification for improved staining and reliability L.C.L. Silveira 1 and V.H. Perry 2 I Departamento de Fisiologia, Universidade Federal do Pard, Bel~m, Pard (Brazil), and : Department of Experimental P~wcholo~,, University of Oxford, Oxford (U.K,) (Received 5 September 1989) (Revised version received 17 January 1990) (Accepted 25 February 1990)
Key words: Neurofilaments; Silver stain; Retina ganglion cells; Skin; Cutaneous receptors A simple modification of a reduced silver-stain for neurofilaments is described. Using neutral pH, perfusion fixation, and heat treatment during postfixation this method greatly improves the quality and reliability of staining of ganglion cells in retinal wholemounts from primate and other species. In addition it permits the selective staining of some cell populations thought to be refractory to silver stains. The method also stains cutaneous receptors in thick sections of the glabrous skin.
Introduction
The use of silver staining methods for visualizing neurofilament containing cell populations in the retina has a long history and was recently reviewed (Wassle et al., 1978). These methods have provided an important tool, for rather than staining individual cells they have provided valuable information about cell populations, how they are distributed and how their morphology may vary across a retina not only in normal material but also following experimental manipulations (W~issle et al., 1978, 1981; Vaney et al., 1981, 1988; Eysel et al., 1985). Although the introduction of monoclonal antibodies has replaced the use of such silver-stains under some conditions (Dr~iger et al., 1984) it is not always possible to obtain material in a form suitable for immunocyCorrespondence: Prof. Luiz Carlos de Lima Silveira, Universidade Federal do Parfi, Centro de Ci~ncias Biol6gicas, Departamento de Fisiologia, 66.059 Belrm, Par~, Brazil. Phone: (091) 229-2088 ext. 570.
tochemistry and this is particularly true of material from valuable species such as monkey and man. Neurofibrillar staining of ganglion cells in the primate retina is difficult. It readily stains the fibres (Vrabec, 1966) but yields only a few poorly stained large cells in the peripheral retina (Peichl et al., 1987 and for earlier references). We were particularly interested to develop a method that would allow us to stain the ganglion cells which project to the magnocellular layers of the dorsal lateral geniculate nucleus (M-ganglion cells) in the monkey retina in a reliable fashion, since the role of these cells in vision is of much interest (Shapley and Perry, 1986). These cells in primate retina have also been called A ganglion cells (Leventhal et al., 1981), P-alpha ganglion cells (Perry et al., 1984), and parasol ganglion cells (Polyak, 1941; Rodieck et al., 1985). Experimental models for depleting the retina of the other major class of ganglion cell, the P-cells have also been recently introduced (Eskin et al., 1985; Eskin and Merigan, 1986). The quantitative analysis of the M-ganglion cell population selectively stained by the present
0165-0270/90/$03.50 © 1990 Elsevier Science Publishers B.V, (Biomedical Division)
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method is currently in preparation (Silveira and Perry, unpublished results). It is well known that many reduced silver methods work better when the tissue is fixed for prolonged periods of time. The chemical reactions which produce the selectivity of these stains are poorly understood and it is not known why this should be the case. We reasoned that it might be possible to produce the same or similar chemical changes more rapidly by heat treatment of the fixed retina and, at the same time, allow better control of the stain selectivity. The method we have finally evolved not only allows us to stain the M-ganglion cells of the primate with a high degree of reliability but greatly improves staining in other species and reveals populations of cells thought to be refractory to silver stains. We have also examined whether this stain may be of more general use by the application of the method to glabrous skin of the primate.
Materials and methods The retinae and samples of skin obtained for these studies came from animals used in physiological, anatomical and behavioral experiments not connected with the present investigation; Animals were anaesthetized with a lethal dose of anaesthetic and perfused transcardially with 0.9% NaC1 solution followed by the fixative solution. This consisted of 10% of a stock 37% formaldehyde solution (BDH) in 0.9% NaC1 solution, which had been stored over marble chips (BDH). We have used marble chips instead of calcium carbonate because the former keeps the pH of the fixative solution in the range of 7.1-7.8, instead of 5.5-6.5. As it is mentioned in the step 2 of the procedures, the pH decreases during the heat treatment. So it is necessary to start the fixation with a slightly higher pH, otherwise the staining will be inhibited. Also our experience is that the calcium carbonate makes the retina brittle and more difficult to handle. The animals used include primates
(Macaca mulatta, Macaca fascicularis, Macaca nemestrina, and Cebus apella ), carnivores (domestic ca0, rodents (rat, agouti, capybara and paca), and lagomorphs (rabbit).
Retinae were dissected from the eye cup within a few hours of fixation and placed in a bottle with the same fixative and several marble chips per 50 ml of solution. Samples of glabrous skin from fingers of Macaca mulatta were stored in the same way. The period of fixation prior to processing varied from 2 weeks to 1 year with no consistent differences.
Procedure The method after steps 1 and 2 is the same as that of Boycott and Peichl (1981) except for minor details.
Retina (1) The retina was flattened on a clean glass plate by making cuts around its circumference. The vitreous humour was carefully removed by holding the retina with a large brush and by brushing the vitreous to the periphery of the retina with a smaller brush. Too much vitreous left on the retina will impair the stain. A small amount of vitreous may be left and this can be removed at the end of the staining. It is particularly important at this stage not to let the retina dry by evaporation as this will inhibit staining. (2) The retina, in the fixative solution with a few marble chips in a closed container, was placed in a 60 °C oven for 24 h. Measurement of the pH prior to heat treatment and immediately following treatment should show a fall of 0.5-1 log unit. (3) Dehydrate through graded alcohols, 30%. 50%. 70%. 90%. 100% (twice), alcohol/xylene (1 : 1), xylene 100%, 5 min in each and finally into xylene 100% for 24 h. (4) Rehydrate through graded alcohols, ending in 0.9% NaCI solution. Saline solution was used instead of distilled water in this step because the latter causes expansion of the retina that becomes fragile and difficult to handle. Saline solution keeps the retina the same size as after step 2. After at least 15 rain in saline solution the retina was mounted fiat, ganglion cell side uppermost, on a slide coated with Fol's solution (Stone, i981). Cleaned slides were dipped and dried at 37 °C for 2 h prior to use and provided very good adhesion.
13 (5) The flattened retina was covered with filter paper (Whatmans No. 1) and another glass side laid on top. The sandwich was placed in a solution (pH 7.2) of 1 part stock 37% formaldehyde solution which had been stored on calcium carbonate to 9 parts absolute ethanol. The sandwich was left overnight. (6) The sandwich was undone and the retina rehydrated diluting the alcohol/formaldehyde solution with a solution (pH 5.5-6.5) of 1 part stock 37% formaldehyde solution stored on calcium carbonate to 9 parts of distilled water. (7) The slide was blotted to remove most of the liquid and placed in a 20% silver nitrate solution for 30 rain on a gently oscillating table. (8) The slide was removed from the silver nitrate solution, the liquid allowed to drain off and placed in 1 part pyridine/formaldehyde solution to 2 parts tap water. The retina was left in this solution until it becomes milky. Three changes in a period of 5 min were sufficent. Pyridine/ formaldehyde solution is analytical grade pyridine 5 parts, formaldehyde 25 parts, distilled water 70 parts. (9) Just prior to the beginning of the developing procedure a silver diamine solution was made up. To 5 ml 20% silver nitrate was added 0.880 ammonia solution drop by drop with continual shaking until the initially formed brown precipitate had just dissolved. This solution was diluted to 20 ml and 10 drops of 0.880 ammonia solution were added. The retina was placed in this silver diamine solution and the reaction monitored under the microscope but at the same time preventing the evaporation of the ammonia by only removing the lid for short periods. The staining is progressive with the axons appearing first followed by the cell bodies and their processes. (10) The reaction was stopped by placing the retina in 1 : 1 ammonia solution and distilled water for 1 min. (11) Washing in distilled water for 1 min. (12) Fixation in 1% sodium thiosulphate for 5 rain. (13) Washing in several changes of distilled water. If vitreous still remained on the surface of
the retina it can be removed at this stage or during dehydration. (14) Dehydration, clearing and coverslipping in the usual fashion. Skin The dermis and epidermis were dissected free from the other tissues and placed in 10% f o r m a l / saline with marble chips. The closed bottle was placed in a 6 0 ° C oven for 24-36 h. The tissue was then transferred to the same fixative containing 20% sucrose, One or 2 days later, when the tissue was well impregnated with the sucrose, it was embedded in OCT embedding compound (Lamb). Sections 70-100 /~m thick were cut into 10% formal/saline over marble chips. The sections were dehydrated through graded alcohols as described above and placed in xylene for 24 h. The sections were then rehydrated and staining proceeded as above from step 7. To facilitate easy handling of the sections all staining was done in 7-cm crystallizing dishes with small baskets made from perspex and nylon netting so that the sections could be kept in serial order. Pins placed through the tissue served as alignment markers.
Results
The method of Boycott and Peichl (1981) provided an important starting point for our method which differs from theirs in only a few but essential steps. The original method developed for staining the neurofilament containing cells of the cat retina used fixation by immersion with formaldehyde stored over lithium carbonate to give a pH of 8-9. In their experience this protocol produced poor staining of neurofibrillar containing ganglion cells in the primate and rat retinae (Peichl et al., 1987) and this has also been our experience. Fixation by perfusion with formaldehyde stored over marble chips, pH 7.1-7.8, improved the quality of staining. We found that immersion fixation of primate retinae gave non-uniform staining and poor resolution of fine processes. However, it was only when perfusion with this fixative was combined with treatment in a 60 °C oven for 24 h that
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Fig. 2. Neurofibrillar-stained wholemounts of two macaque monkey retinae (Macaca mulatta). Scale bars = 100 /~m. A - B : giant displaced ganglion cell with very large cell body and dendritic tree. It was found 6 mm temporal to the fovea centralis. The axon (arrow) branches extensively across a wide area of the retina. This cell type stains as intensely as the alpha-like ganglion cells of non-primate mammals. C: axon from the same cell seen in A - B branching 1.8 mm from the cell body. D: efferent fibre branching 1 mm after emerging from the optic disk.
the quality of staining and reproducibility were dramatically enhanced. A variety of heat treatment protocols were tried, for example 3 7 ° C for periods varying from a few days to 3 months but
as yet none has given consistent or as satisfactory results as with the present procedure. A major consequence of the heat treatment is that it greatly improves the selectivity of the stain,
Fig. 1. Neurofibrillar-stained wholemount of macaque monkey retina (Macaca fascicularis). M-ganglion cell bodies, axons, and primary dendrites are intensely stained. Some of the high order dendritic branches are also stained. A: low power view of the ganglion cell layer. Mid-periphery, temporal retinal region. Focus on the nucleoli of 3 inner M-ganglion cells. Two outer M-ganglion cell bodies are placed slightly external to the inner M-ganglion cell bodies and are out of focus in this micrograph. Scale bar = 100 /~m. B: outer M-ganglion cell of the temporal retinal region. Focus on the dendritic tree, close to the inner nuclear layer. Scale bar = 50/zm.
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suppresses the background staining of the end feet of the Muller cells and reduces the staining of nuclei of the inner and outer nuclear layers. A common problem with neurofibrillar stains applied to the primate retina is the dark background and non-uniform staining. This method allows uniform staining of a select population of ganglion cells but the entire dendritic tree was rarely stained (Fig. 1). In the primate retina, close to the fovea, the density of cells with neurofilaments in their cytoplasm increased but the cell bodies were smaller and the extent of the dendritic staining decreased. At an eccentricity of about 2 mm from the fovea where the ganglion cell layer becomes multilayered, the majority of these cells lay deep within the ganglion cell layer and becomes increasingly difficult to resolve. The staining was consistent between individual retinae. In a number of these retinae we have identified occasional displaced ganglion cells with very large dendritic trees. Their axons enter the optic fibre layer but in addition they have axon collaterals which are extensively branched across a wide area of the retina (Fig. 2A-C). Other extensively branched axons were seen to originate from the optic nerve head and were presumably rare centrifugal fibres (Fig. 2D). When applied to the retina of other species the quality of staining was also very good. In the agouti cells they were exceptionally well stained, which with previous methods could only be resolved against a very dark background (Fig. 3). In addition to the neurofibrillar ganglion cells, horizontal cells, coronate amacrine cells, and processes of long range amacrine cells, which have been reported by others (Wassle et al., 1978, 1981; Peichl et al., 1987; Vaney et al., 1981, 1988), we have succeeded in staining a number of cell types that were thought to be refractory. For example, the alpha-like ganglion cells of the rat exist in 2 subtypes branching in either the outer part of the inner plexiform layer or the middle of the plexi-
form layer but the cells branching in the outer part are difficult to stain with previous neurofibrillar stains (Peichl et al., 1987). With this method it is possible to stain these cells (Fig. 4A-C). In the adult rat we also stained neurofilament containing processes in the outer plexiform layer but a description of them will be the subject of a future publication. To examine whether this method might be of value in other tissues we stained horizontal thick sections of glabrous skin of the primate (Fig. 5A). The staining of Meissner's corpuscles was of good quality. Within the corpuscle the nerve endings can be seen as heavily stained coiled processes (Fig. 5B). The uniformity of the staining in thick sections would readily allow reconstruction of their density over large areas of tissues.
Discussion
The staining of neurofibrillar containing elements in the primate retina by this method was reliable and superior to that obtained using other methods. The effect of the heat treatment is not known and we are unaware of any previous systematic investigations into the possibility of using this approach. The only comment we are aware of is by Da Fano who having observed that better staining was produced by prolonged fixation attempted to produce rapid fixation, and who states "10 to 20 per cent formalin at 3 7 ° C gave bad results" (p. 531, in Gattenby and Beams, 1950). It should be noted that initial fixation in a warm fixative may be rather different from fixing at room temperature followed by heat treatment. The chemical change produced by the heat treatment is stable for some time since for both skin and retina we have on several occasions left the tissue following heat treatment for several days prior to subsequent staining with good results.
Fig. 3. NeurofibriUar-stained wholemount of agouti retina (Dasyprocta aguti) showing an i n n e r / o u t e r pair of alpha-like ganglion cells. They are the 2 large cells in the field. In addition to the alpha-like ganglion cells other ganglion cells (arrow) and displaced amacrine cells are also stained. Scale bars = 100 ~m. A: focus on the dendritic tree of the non-alpha ganglion cell. B: focus on the dendritic tree of the inner alpha-like ganglion cell.
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J Fig. 4. Neurofibrillar-stained wholemount of rat retina showing an t u n e r / o u t e r pair of alpha-like ganglion cells. Scale bar = 60 # m valid for all the micrographs in this figure. A: focus on the nucleolus of the inner alpha-like ganglion cell. B: focus on the dendritic tree of the inner alpha-like ganglion cell. C: focus on the dendritic tree of the outer alpha-like ganglion cell.
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Fig. 5. A: horizontal skin section stained by the neurofibrillar method. Regularly spaced Meissner corpuscles are seen lying in rows and the nerve fibres within them are heavily stained. Scale bar = 200 tim. B: high-power view, showing an example of a convoluted nerve ending. Scale bar = 50/~m.
20 T h e i m p r o v e m e n t in the q u a l i t y of staining was f o u n d not o n l y with the p r i m a t e r e t i n a b u t in all the species we have examined. It is of interest that the b a c k g r o u n d we o b s e r v e d w i t h o u t heat treatm e n t was largely due to staining of the e n d feet of the Muller cells. M u l l e r cells are k n o w n to c o n t a i n vimentin, a n o t h e r i n t e r m e d i a t e f i l a m e n t protein, in some species (Shaw a n d W e b e r , 1983; Dr~iger, 1983). W e have n o t seen staining of retinal astrocytes which c o n t a i n the i n t e r m e d i a t e f i l a m e n t glial fibrillary acidic p r o t e i n (see Schnitzer, 1988, for review). T h e heat t r e a t m e n t a p p e a r s to influence the affinity of silver for p a r t i c u l a r i n t e r m e d i a t e filaments a n d it m i g h t be of s o m e interest to s t u d y this p h e n o m e n o n in m o r e detail. T h e presence o f b r a n c h e d a x o n like structures within the i n n e r p l e x i f o r m layer of the m a m m a l i a n r e t i n a has p r e v i o u s l y b e e n o b s e r v e d b y o t h e r a u t h o r s a n d it has b e e n suggested that these a x o n s m a y be of centrifugal origin ( H o n r u b i a a n d Elliot, 1970; D r a g e r et al., 1984). However, there is a n o t h e r source of i n t r a r e t i n a l axons, occasional ganglion cells with an i n t r a r e t i n a l axon have been previously r e p o r t e d in the cat a n d o t h e r species (see Dacey, 1985, a n d for earlier references) b u t n o t in m o n k e y retina. T h e r a r i t y of these cells in the m o n k e y r e t i n a suggests to us that these cells are a d e v e l o p m e n t a l a b n o r m a l i t y . F o r the s t u d y of select p o p u l a t i o n s o f r e t i n a l cells a n d c u t a n e o u s r e c e p t o r s this m e t h o d m a y p r o v i d e a v a l u a b l e a d j u n c t to the wide range o f m o d e r n a n a t o m i c a l techniques used to s t u d y b o t h n o r m a l a n d e x p e r i m e n t a l material. W i t h further investigation it m i g h t b e p o s s i b l e to o b t a i n even b e t t e r c o n t r o l of the selectivity.
Acknowledgements This w o r k was s u p p o r t e d b y research g r a n t s f r o m C N P q / T h e R o y a l Society (No. 9 1 0 1 0 8 / 8 8 . 9 a n d No. 4 0 1 8 0 8 / 8 8 ) , F I N E P / F A D E S P No. 4.3.87.0308.00, T h e W e l l c o m e Trust, a n d U F P a P R O P E S P . V.H.P. is a W e l l c o m e Senior R e s e a r c h Fellow. L.C.L.S. has a research fellowship f r o m C N P q No. 302216/82. W e wish to t h a n k C. H e a l e y - Y o r k e for excellent technical assistance, a n d
S.M.A. d e L i m a for r e s e a r c h assistance. A m a z o n a n i m a l s used in this w o r k were p r o v i d e d by MPEG, CENP, and Eletronorte.
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21 Vaney, D,I., Peichl, L. and Boycott, B.B. (1988) Neurofibrillar long range amacrine cells in mammalian retina. Proc. R. Soc. Lond. B, 235: 203-219. Vrabec, M.D. (1966) The temporal raphe of the human retina. Am. J. Ophthalmol., 62: 926-938. Wassle, H., Peichl, L, and Boycott, B.B. (1978) Topography of
horizontal cells in the retina of the domestic cat. Proc. R. Soc. Lond. B, 203: 269-291. Wassle, H., Peichl, L. and Boycott, B.B. (1981) Morphology and topography of on- and off-alpha ceils in the cat retina. Proc. R. Soc. Lond. B, 212: 157-175.