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Effects of aldehydes and organic solvents on Concanavalin A binding sites in cerebellar tissue sections
Neuroscience Letters, 12 (1979) 355--360 © Elsevier/North-Holland Scientific Publishers Ltd.
355
EFFECTS OF ALDEHYDES AND ORGANIC SOLVENTS ON CONCANAVALIN A BINDING SITES IN CEREBELLAR TISSUE SECTIONS
A N N E T T E F I J C H T B A U E R and M E L I T T A S C H A C H N E R * Department of Neurobiology, Heidelberg University, Im Neuenheimer Feld 320, D-6900 Heidelberg (F.R.G.)
(Received January 19th, 1979) (Accepted January 20th, 1979)
SUMMARY
Fresh frozen cerebellar sections of adult mice treated with aldehydes and organic solvents reveal differences in detectability of Concanavalin A (Con A) binding sites. While fluorescein coupled Con A shows intense labeling of synaptic glomeruli and granule cell bodies under all conditions, the molecular layer labels intensely after treatment with paraformaldehyde, glutaraldehyde, acetone, ethanol and butanol. Complete loss of staining in molecular and granular layers and substantial increase in white matter labeling occurs after chloroform-methanol treatment. Except for glutaraldehyde treated sections, all labeling is specifically inhibited by methyl~-D-mannoside, but not by galactose.
The histological characterization and localization of nervous system constituents can be achieved through the utilization of visually tagged reagents with defined binding specificities: antibodies, lectins and toxins. Histological sections offer the unique advantage over biochemical studies to correlate binding site with cellular and subcellular distribution. Since cells of the nervous system are organized domains of structural and functional properties (for instance, cell body, axon, dendrite, pre- and post-synaptic site), a topographical analysis, particularly during development, seems important. In general, histological procedures depend on tissue fixation which signifies the denaturation of proteins. This can be achieved by freezing and thawing and treatment with aldehydes, acids or organic solvents. However, each of these treatments affects the molecular properties of the tissue in a different way. Some treatments may even destroy the specific binding sites. On the other hand, agents like dimethylsulfoxide (DMSO) or detergents may enhance the penetration of high molecular weight compounds [3] so *To w h o m allcorrespondence should be sent.
356 that they may reach their target molecules more easily. Another factor which can affect the detection of tissue constituents is the masking of binding sites, for instance by a lipid rich environment in myelinated structures [4]. In addition, organic solvents have been introduced to reduce nonspecific background staining which is a particular problem in immunohistology [ 1 ]. To evaluate these factors in a systematic manner we have chosen the plant lectin Concanavalin A (Con A) [2] to study the detectability of mannose containing carbohydrates in tissue sections of adult mouse cerebellum. Brains from adult C57B1/6J mice bred in our colony were fresh frozen on dry ice, mounted in OTC (Miles) and cut in the sagittal plane in a Cryocut (Jung, Nussloch, F.R.G.) at - 1 2 ° C . Sections (10 ~m thick) were melted onto glass slides and allowed to dry for 1 hr at room temperature. All further manipulations were carried out at 0°C. Sections in large Petri dishes with a humidified atmosphere, were exposed to various agent for 15 min: 5% dimethylsulfoxide (DMSO, Merck) in phosphate buffered saline {PBS) (pH 7.3); 4% paraformaldehyde (Merck)and 0.05% glutaraldehyde (Serva), also in PBS; treatment with ethanol and acetone (Merck) occurred in graded steps (incubation for 3 rain each in 30%, 50%, 70% and 100% and back to 70%, 50%, 30% and PBS; 100% N-butanol and chloroform methanol at 2 : 1 (v/v). After three washes with PBS (3 min each) the sections are treated with 0.5% bovine serum albumin (BSA) in PBS for 10 min at 0°C to saturate unspecific binding sites for proteins and inactivate reactive aldehyde groups. Sections were again washed two times. Fluorescein conjugated Concanavalin A (Con. A-FITC) at a concentration of 10 pg/ml of PBS is then applied to the section for 15 min (see ref. 2 for details). Sections are then washed three times, m o u n t e d in glycerol-PBS (1 : 1, v/v) and inspected with a Zeiss fluorescence microscope with epi-illumination. To prove the specificity of the Con A binding reaction hapten sugar inhibition was carried out with methyl-a-D-mannoside at concentrations of 500, 50 and 5 mM. Inhibition was performed by incubating Con A and sugar for 10 min prior to application to the tissue section. At concentrations of 500 and 50 mM inhibition of Con A-FITC binding was complete. At 50 mM concentrations binding was reduced but still detectable, while the pattern of labeling was identical to the noninhibited Con A pattern. Inhibition experiments were therefore carried out at concentrations of 50--500 mM. To prove specificity of the hapten sugar inhibition reaction galactose was used at the same concentrations. Binding of Con A-FITC to sagittai sections of fresh frozen cerebellum of adult C57B1/6J mice showed distinct, reproducible labeling patterns. To assure the specificity of the reaction non-specific protein binding sites in sections were saturated by incubation with 0.5% BSA in PBS prior to treatment with Con A-FITC (Hatten et al., unpublished data). Under such conditions the distribution of Con A binding sites is characterized by a predominant detectability in the granular layer (GL), with an intense labeling of the outlines of granular cell (GC) bodies and of synaptic glomeruli (SG, Fig. 3).
357
Fig. 1--12. Con A-FITC binding to cerebellar tissue sections after various pretreatments: BSA (1--4); DMSO and BSA (5--8); paraforrnaldehyde and BSA (9--12). Con A binding inhibited by methyl-a-D-mannoside (4,8,12). Magnifications: x 100 (1,5,9); × 250 (2,6,10): × 600 (3,4,7,8,11,12).
358
Fig. 13--20. Con A-FITC binding to cerebellar tissue sections after various pretreatments: glutaraldehyde and BSA (13,14); N-butanol and BSA (15); acetone and BSA ~16_17)~ chloroform--methanol and BSA (18--20). Methyl-a-D-rnannoside was added (14,20) to test for specificity of Con A binding. Magnifications: x l 0 0 (18); × 250 (13--16,19,20); × 600 (17).
359 While Con A binding sites are relatively difficult to discern in the white matter (WM) the molecular layer (ML)shows a faint binding reaction (Figs. 1 and 2) as has been observed before [5]. The staining reaction is specific, since it can be inhibited by methyl-~-D-mannoside but not by galactose (Fig. 4). Preincubation of the sections with 5% DMSO in PBS prior to treatment with BSA and Con A-FITC results in a labeling pattern which is identical to the one obtained without DMSO treatment and reveals a slightly decreased binding activity, particularly in white matter (Figs. 5 and 6). Granule cell bodies are outlined by a fluorescent rim (Fig. 7). The staining reaction is specific (Fig. 8). Treatment with 4% paraformaldehyde results in an increase of binding sites in the molecular layer relative to granular layer and white matter (Figs. 9 and 10). The overall staining intensity is also increased when compared to sections treated with BSA and DMSO. The pattern of labeling in the granular layer (Fig. 11) is identical to the labeling pattern observed in sections without and with DMSO treatment. The staining reaction is specific (Fig. 12). A substantial increase in overall yellow, but not green-yellow labeling intensity is observed after glutaraldehyde treatment (Fig. 13). The labeling pattern is comparable to the one obtained with paraformaldehyde. This reaction is not specifically inhibitable by hapten sugar, even at the highest concentration (500 mM) (Fig. 14). N-Butanol treatment results in a specifically inhibitable labeling pattern similar to the one observed after paraformaldehyde treatment (Fig. 15). Treatment with acetone (Figs. 16 and 17) and ethanol (not shown) increases Con A binding sites in the white matter relative to molecular and granular layers (Fig. 16). The Con A binding reaction is again specifically inhibitable b y hapten sugar. Most striking is the loss of Con A binding sites in granular, molecular and Purkinje cell layers (PCL) and a drastic increase of binding sites in white matter after chloroform--methanol treatment (Figs. 18 and 19). This binding reaction is specific (Fig. 20). The present study shows that the detectability of Con A binding sites in adult mouse cerebellum depends on the treatment of the tissue section prior to application of Con A-FITC. While a predominant labeling of the granular layer is observed in sections not treated or treated with DMSO the molecular layer labels heavily after fixation with paraformaldehyde and butanol. White matter becomes increasingly dominant after incubation with acetone and ethanol. Upon treatment with chloroform--methanol white matter remains as solely labeled cerebellar structure. This binding is specific, since in contrast to other studies [ 5] it is inhibitable by methyl-a-D-mannose. While differences in labeling pattern have been observed in our experiments it is difficult to assess why the binding sites become more or less detectable under various fixation procedures. It is likely, however, that unmasking of Con A binding sites in white matter is due to extraction of lipid components
360 as it has b e e n o b s e r v e d for m y e l i n basic p r o t e i n [4]. In a d d i t i o n , d i f f e r e n t shades o f r e a c t i v i t y o f t h e m o l e c u l a r layer ranging f r o m c o m p l e t e n e g a t i v i t y to s t r o n g p o s i t i v i t y c o u l d be e x p l a i n e d b y selective d i s t r i b u t i o n or e x t r a c t i o n b y organic solvents o f lipid rich Con A binding sites. I n c r e a s e d accessability c o u l d a c c o u n t f o r high staining o f t h e m o l e c u l a r layer after, for instance, p a r a f e r m a l d e h y d e t r e a t m e n t . Finally, o u r s t u d y shows t h a t e x t r e m e c a u t i o n has to be t a k e n w h e n g l u t a r a l d e h y d e fixed sections are e x p o s e d to Con A since strong n o n - s p e c i f i c r e a c t i v i t y results. T h e p r e s e n t s t u d y e m p h a s i z e s the i m p o r t a n c e o f c o n t r o l l e d t r e a t m e n t o f histological s p e c i m e n s in binding studies and shows t h a t t h e d e t e c t a b i l i t y of pariciular binding sites m a y d e p e n d solely o n these t r e a t m e n t s . ACKNOWLEDGEMENTS This w o r k was s u p p o r t e d b y D e u t s c h e F o r s c h u n g s g e m e i n s c h a f t , S t i f t u n g V o l k s w a g e n w e r k , G e m e i n n i i t z i g e H e r t i e - S t i f t u n g and N a t i o n a l I n s t i t u t e s o f Health. REFERENCES 1 Hartmann, B.K., Zide, D. and Udenfriend, S., The use of dopamine t3-hydroxylase as a marker for the central noradrenergic nervous system in the rat brain, Proc. nat. Acad. Sci. (Wash.), 69 (1972) 2722--2726. 2 Lis, H. and Sharon, N., The biochemistry of plant lectins (phytohemagglutinius), Ann. Rev. Biochem., 42 (1973) 541--574. 3 Schachner, M., Hedley-Whyte, E.T., Hsu, D.W., Schoonmaker, G. and Bignami, A., Ultrastructural localization of glial fibrillary acidic protein in mouse cerebellum by immunperoxidase labeling, J. Cell Biol., 75 (1977) 67--73. 4 Sternberger, N.H., Itoyama, Y., Kies, M.W. and Webster, H. DEF., Myelin basic protein demonstrated immunocytochemically in oligodendroglia prior to myelin sheath formation, Proc. nat. Acad. Sci. (Wash), 75 (1978) 2521--2524. 5 Zanetta, J.P., Roussel, G., Ghandour, M.S., Vincendon, G. and Gombos, G., Postnatal development of rat cerebellum: massive and transient accumulation of Con A binding glycoproteins in parallel fiber axolemma, Brain Res., 142 (1978) 301--319.
355
EFFECTS OF ALDEHYDES AND ORGANIC SOLVENTS ON CONCANAVALIN A BINDING SITES IN CEREBELLAR TISSUE SECTIONS
A N N E T T E F I J C H T B A U E R and M E L I T T A S C H A C H N E R * Department of Neurobiology, Heidelberg University, Im Neuenheimer Feld 320, D-6900 Heidelberg (F.R.G.)
(Received January 19th, 1979) (Accepted January 20th, 1979)
SUMMARY
Fresh frozen cerebellar sections of adult mice treated with aldehydes and organic solvents reveal differences in detectability of Concanavalin A (Con A) binding sites. While fluorescein coupled Con A shows intense labeling of synaptic glomeruli and granule cell bodies under all conditions, the molecular layer labels intensely after treatment with paraformaldehyde, glutaraldehyde, acetone, ethanol and butanol. Complete loss of staining in molecular and granular layers and substantial increase in white matter labeling occurs after chloroform-methanol treatment. Except for glutaraldehyde treated sections, all labeling is specifically inhibited by methyl~-D-mannoside, but not by galactose.
The histological characterization and localization of nervous system constituents can be achieved through the utilization of visually tagged reagents with defined binding specificities: antibodies, lectins and toxins. Histological sections offer the unique advantage over biochemical studies to correlate binding site with cellular and subcellular distribution. Since cells of the nervous system are organized domains of structural and functional properties (for instance, cell body, axon, dendrite, pre- and post-synaptic site), a topographical analysis, particularly during development, seems important. In general, histological procedures depend on tissue fixation which signifies the denaturation of proteins. This can be achieved by freezing and thawing and treatment with aldehydes, acids or organic solvents. However, each of these treatments affects the molecular properties of the tissue in a different way. Some treatments may even destroy the specific binding sites. On the other hand, agents like dimethylsulfoxide (DMSO) or detergents may enhance the penetration of high molecular weight compounds [3] so *To w h o m allcorrespondence should be sent.
356 that they may reach their target molecules more easily. Another factor which can affect the detection of tissue constituents is the masking of binding sites, for instance by a lipid rich environment in myelinated structures [4]. In addition, organic solvents have been introduced to reduce nonspecific background staining which is a particular problem in immunohistology [ 1 ]. To evaluate these factors in a systematic manner we have chosen the plant lectin Concanavalin A (Con A) [2] to study the detectability of mannose containing carbohydrates in tissue sections of adult mouse cerebellum. Brains from adult C57B1/6J mice bred in our colony were fresh frozen on dry ice, mounted in OTC (Miles) and cut in the sagittal plane in a Cryocut (Jung, Nussloch, F.R.G.) at - 1 2 ° C . Sections (10 ~m thick) were melted onto glass slides and allowed to dry for 1 hr at room temperature. All further manipulations were carried out at 0°C. Sections in large Petri dishes with a humidified atmosphere, were exposed to various agent for 15 min: 5% dimethylsulfoxide (DMSO, Merck) in phosphate buffered saline {PBS) (pH 7.3); 4% paraformaldehyde (Merck)and 0.05% glutaraldehyde (Serva), also in PBS; treatment with ethanol and acetone (Merck) occurred in graded steps (incubation for 3 rain each in 30%, 50%, 70% and 100% and back to 70%, 50%, 30% and PBS; 100% N-butanol and chloroform methanol at 2 : 1 (v/v). After three washes with PBS (3 min each) the sections are treated with 0.5% bovine serum albumin (BSA) in PBS for 10 min at 0°C to saturate unspecific binding sites for proteins and inactivate reactive aldehyde groups. Sections were again washed two times. Fluorescein conjugated Concanavalin A (Con. A-FITC) at a concentration of 10 pg/ml of PBS is then applied to the section for 15 min (see ref. 2 for details). Sections are then washed three times, m o u n t e d in glycerol-PBS (1 : 1, v/v) and inspected with a Zeiss fluorescence microscope with epi-illumination. To prove the specificity of the Con A binding reaction hapten sugar inhibition was carried out with methyl-a-D-mannoside at concentrations of 500, 50 and 5 mM. Inhibition was performed by incubating Con A and sugar for 10 min prior to application to the tissue section. At concentrations of 500 and 50 mM inhibition of Con A-FITC binding was complete. At 50 mM concentrations binding was reduced but still detectable, while the pattern of labeling was identical to the noninhibited Con A pattern. Inhibition experiments were therefore carried out at concentrations of 50--500 mM. To prove specificity of the hapten sugar inhibition reaction galactose was used at the same concentrations. Binding of Con A-FITC to sagittai sections of fresh frozen cerebellum of adult C57B1/6J mice showed distinct, reproducible labeling patterns. To assure the specificity of the reaction non-specific protein binding sites in sections were saturated by incubation with 0.5% BSA in PBS prior to treatment with Con A-FITC (Hatten et al., unpublished data). Under such conditions the distribution of Con A binding sites is characterized by a predominant detectability in the granular layer (GL), with an intense labeling of the outlines of granular cell (GC) bodies and of synaptic glomeruli (SG, Fig. 3).
357
Fig. 1--12. Con A-FITC binding to cerebellar tissue sections after various pretreatments: BSA (1--4); DMSO and BSA (5--8); paraforrnaldehyde and BSA (9--12). Con A binding inhibited by methyl-a-D-mannoside (4,8,12). Magnifications: x 100 (1,5,9); × 250 (2,6,10): × 600 (3,4,7,8,11,12).
358
Fig. 13--20. Con A-FITC binding to cerebellar tissue sections after various pretreatments: glutaraldehyde and BSA (13,14); N-butanol and BSA (15); acetone and BSA ~16_17)~ chloroform--methanol and BSA (18--20). Methyl-a-D-rnannoside was added (14,20) to test for specificity of Con A binding. Magnifications: x l 0 0 (18); × 250 (13--16,19,20); × 600 (17).
359 While Con A binding sites are relatively difficult to discern in the white matter (WM) the molecular layer (ML)shows a faint binding reaction (Figs. 1 and 2) as has been observed before [5]. The staining reaction is specific, since it can be inhibited by methyl-~-D-mannoside but not by galactose (Fig. 4). Preincubation of the sections with 5% DMSO in PBS prior to treatment with BSA and Con A-FITC results in a labeling pattern which is identical to the one obtained without DMSO treatment and reveals a slightly decreased binding activity, particularly in white matter (Figs. 5 and 6). Granule cell bodies are outlined by a fluorescent rim (Fig. 7). The staining reaction is specific (Fig. 8). Treatment with 4% paraformaldehyde results in an increase of binding sites in the molecular layer relative to granular layer and white matter (Figs. 9 and 10). The overall staining intensity is also increased when compared to sections treated with BSA and DMSO. The pattern of labeling in the granular layer (Fig. 11) is identical to the labeling pattern observed in sections without and with DMSO treatment. The staining reaction is specific (Fig. 12). A substantial increase in overall yellow, but not green-yellow labeling intensity is observed after glutaraldehyde treatment (Fig. 13). The labeling pattern is comparable to the one obtained with paraformaldehyde. This reaction is not specifically inhibitable by hapten sugar, even at the highest concentration (500 mM) (Fig. 14). N-Butanol treatment results in a specifically inhibitable labeling pattern similar to the one observed after paraformaldehyde treatment (Fig. 15). Treatment with acetone (Figs. 16 and 17) and ethanol (not shown) increases Con A binding sites in the white matter relative to molecular and granular layers (Fig. 16). The Con A binding reaction is again specifically inhibitable b y hapten sugar. Most striking is the loss of Con A binding sites in granular, molecular and Purkinje cell layers (PCL) and a drastic increase of binding sites in white matter after chloroform--methanol treatment (Figs. 18 and 19). This binding reaction is specific (Fig. 20). The present study shows that the detectability of Con A binding sites in adult mouse cerebellum depends on the treatment of the tissue section prior to application of Con A-FITC. While a predominant labeling of the granular layer is observed in sections not treated or treated with DMSO the molecular layer labels heavily after fixation with paraformaldehyde and butanol. White matter becomes increasingly dominant after incubation with acetone and ethanol. Upon treatment with chloroform--methanol white matter remains as solely labeled cerebellar structure. This binding is specific, since in contrast to other studies [ 5] it is inhibitable by methyl-a-D-mannose. While differences in labeling pattern have been observed in our experiments it is difficult to assess why the binding sites become more or less detectable under various fixation procedures. It is likely, however, that unmasking of Con A binding sites in white matter is due to extraction of lipid components
360 as it has b e e n o b s e r v e d for m y e l i n basic p r o t e i n [4]. In a d d i t i o n , d i f f e r e n t shades o f r e a c t i v i t y o f t h e m o l e c u l a r layer ranging f r o m c o m p l e t e n e g a t i v i t y to s t r o n g p o s i t i v i t y c o u l d be e x p l a i n e d b y selective d i s t r i b u t i o n or e x t r a c t i o n b y organic solvents o f lipid rich Con A binding sites. I n c r e a s e d accessability c o u l d a c c o u n t f o r high staining o f t h e m o l e c u l a r layer after, for instance, p a r a f e r m a l d e h y d e t r e a t m e n t . Finally, o u r s t u d y shows t h a t e x t r e m e c a u t i o n has to be t a k e n w h e n g l u t a r a l d e h y d e fixed sections are e x p o s e d to Con A since strong n o n - s p e c i f i c r e a c t i v i t y results. T h e p r e s e n t s t u d y e m p h a s i z e s the i m p o r t a n c e o f c o n t r o l l e d t r e a t m e n t o f histological s p e c i m e n s in binding studies and shows t h a t t h e d e t e c t a b i l i t y of pariciular binding sites m a y d e p e n d solely o n these t r e a t m e n t s . ACKNOWLEDGEMENTS This w o r k was s u p p o r t e d b y D e u t s c h e F o r s c h u n g s g e m e i n s c h a f t , S t i f t u n g V o l k s w a g e n w e r k , G e m e i n n i i t z i g e H e r t i e - S t i f t u n g and N a t i o n a l I n s t i t u t e s o f Health. REFERENCES 1 Hartmann, B.K., Zide, D. and Udenfriend, S., The use of dopamine t3-hydroxylase as a marker for the central noradrenergic nervous system in the rat brain, Proc. nat. Acad. Sci. (Wash.), 69 (1972) 2722--2726. 2 Lis, H. and Sharon, N., The biochemistry of plant lectins (phytohemagglutinius), Ann. Rev. Biochem., 42 (1973) 541--574. 3 Schachner, M., Hedley-Whyte, E.T., Hsu, D.W., Schoonmaker, G. and Bignami, A., Ultrastructural localization of glial fibrillary acidic protein in mouse cerebellum by immunperoxidase labeling, J. Cell Biol., 75 (1977) 67--73. 4 Sternberger, N.H., Itoyama, Y., Kies, M.W. and Webster, H. DEF., Myelin basic protein demonstrated immunocytochemically in oligodendroglia prior to myelin sheath formation, Proc. nat. Acad. Sci. (Wash), 75 (1978) 2521--2524. 5 Zanetta, J.P., Roussel, G., Ghandour, M.S., Vincendon, G. and Gombos, G., Postnatal development of rat cerebellum: massive and transient accumulation of Con A binding glycoproteins in parallel fiber axolemma, Brain Res., 142 (1978) 301--319.