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Anti-goldfish glial fibrillary acidic protein (GFAP) recognises astrocytes from rat CNS
Brain Research, 504 (1989) 343-346
343
Elsevier
BRES 23847
Anti-goldfish glial fibrillary acidic protein (GFAP) recognises astrocytes from rat CNS Sara S.A. Shehab, Carole A. Stafford, Shmaiel N. Nona and John R. Cronly-Dillon Developmental Neurobiology Laboratory, Department of Optometry and Vision Sciences, U.M. L S. T., Manchester (U. K. )
(Accepted 22 August 1989) Key words: Glial fibrillary acidic protein; Astrocyte; Immunohistochemistry
A polyclonal antibody to goldfish GFAP recognises, immunohistochemically, astrocyte populations in rat brain, spinal cord and optic nerve. The pattern of staining compares favourablywith that obtained using a polyclonalanti-human GFAP or a monoclonal anti-porcine GFAE These results are consistent with the notion that GFAP is well conserved in vertebrate phylogeny. The intermediate filament cytoskeletal fraction from goldfish brain contains a prominent protein, which has the same electrophoretic mobility as the 51 kDa GFAP isolated similarly from rat brain and spinal cord. We have termend this protein goldfish GFAP 5. An antibody produced in rabbit to the purified goldfish GFAP, which cross-reacts with purified rat GFAP on immunobloP, has been used in this study to localise astrocytes in rat CNS. Norwegian black hooded rats, bred in our laboratories, were used in this study. Tissue from cerebrum, cerebellum, spinal cord and optic nerve was quickly frozen in liquid nitrogen and then cut on a cryostat (-20 °C). Sections (20/~m) were picked up onto gelatincoated slides, dried at room temperature for 1 h and then fixed in eithe[ 4% paraformaldehyde in PBS (pH 7.3) or in acetic acid: alcohol (5:95), for 0.5 h at room temperature. Sections were washed with PBS and then incubated with anti-goldfish GFAP 0:200-1:400)or antihuman GFAP (1:400) in PBS at 4 °C in a humid chamber overnight. This was followed by successive hourly treatments, at room temperature, with swine-anti-rabbit (1:50; Dako) and peroxidase-anti-peroxidase a (PAP, 1:100; Dako). Also used was a monoclonal anti-porcine GFAP (1:100-1:200; ICN). The secondary antibody used was biotinylated sheep anti-mouse IgG (1:50; Amersham), and this was followed by streptavidin biotinylated horseradish peroxidase 3 (1:100; Amersbam). The reaction product in both cases was visualised with 3,3"diaminobenzidine/hydrogen peroxide, before being prepared for observation with a Leitz Diaplan light microscope. For double-labelling studies, sections incubated
with primary antibodies were then incubated with either fluorescein (FITC) conjugated goat-anti-mouse IgG (1:50; Sigma), for the monoclonal anti-GFAP, or rhodamine (TRITC) conjugated goat-anti-rabbit IgG (1:50; Sigma), for the polyclonal anti-goldfish GFAP. All three antibodies used in this study recognised astrocytes in rat brain, spinal cord and optic nerve. In cerebeilar white matter, anti-goldfish GFAP recognised astrocytes of stellate morphology (Fig. la). However, while the density of these positive structures was similar to that observed with anti-human GFAP (Fig. lb) and anti-porcine GFAP (not shown), the intensity of the processes was somewhat weaker. In our original report s, it was shown that Bergmann fibres were also clearly stained with anti-goldfish GFAP; here, we further demonstrate, by the use of double labelling, that all the structures recognised by anti-porcine GFAP are also recognised by anti-goldfish GFAP (Figs. 2 and 3). The staining pattern in the hippocampus also revealed astrocytic structures of stellate morphology, with antigoldfish GFAP (Fig. 4a). Here, both the number of positive structures and their intensity were somewhat less than those observed with either anti-porcine GFAP (Fig. 4b), or with anti-human GFAP (not shown). In white matter of spinal cord, stellate as well as radially oriented 4 (Fig. 5), astrocytes were recognised with anti-goldfish GFAP, while in optic nerve GFAP immunoreactivity was in the form of a network, similar to the staining observed with anti-human GFAP (manuscript in preparation). Preahsorption of goldfish antiGFAP with goldfish GFAP showed no positive imn~luno-
Correspondence: S.N. Nona, Developmental Neurology Lab., Dept. Optometry and Vision Sci., U.M.I.S.T., Manchester M60 1QD,U.K.
0006-8993/891503.50© 1989 Elsevier Science Publishers B.V. (Biomedical Division)
Fig. 1. Sagittal sections of rat cerebellar white matter immunolabelled with antibodies to (a) goldfish GFAP and (b) human GFAP (PAP
method). Bar -- 50 ~m.
l'lg. 2. Sagittal sections of rat cerebellum showing Bergmann fibres double-labelled with antibodies to (a) goldfish GFAP (TRITC) and (b) porcine GFAP (FITC). Arrow indicates piai surface. Bar = 100/~m.
Fig. 3. Sagittal sections of rat cerebellar white matter double-labelled with antibodies to (a) goldfish GFAP (TRITC) and (b) porcine GFAP (FITC). Bar -- 100 ~m.
Fig. 4. Coronal sections of brain hippocampal region immunolabelled with antibodies to (a) goldfish GFAP and (b) porcine GFAP (PAP). Bar = 50/~m.
346
Fig. 5. Cross-section of rat spinal cord immunolabelled with anti-goldfish GFAP (PAP) showing the presence of astrocytes of stellate (arrow) and radial (arrowhead) morphology. Bar = 50/zm.
reactivity (56/~g GFAP per mi diluted antibody, incubated at room temperature for 2 h). The above immunohistochemical results confirm that GFAP is well conserved in vertebrate phylogeny. Previous work had shown that antibodies raised against GFAP from human, shark, chicken and bovine cross-react with GFAP from brain and spinal cord of several higher and lower vertebrates, with only a few exceptions t'2'6. One of the main exceptions was goldfish, where anti-human GFAP failed to recognise GFAP-positive glia in rectum 1 or in optic nerve and tract1'7. However, anti-goldfish GFAP described in the present work has been shown in normal goldfish to strongly label ependymal glia in tectum as well as an array of processes in optic tract,
whilst in optic nerve the labelling is very weak s. The fact that this anti-goldfish GFAP also recognises astrocyte populations in rat CNS, as shown in this study, suggests that it has epitopes not only to goldfish GFAP but also to rat GFAP, although these appear to be less abundant in the latter case. The availability of an anti-GFAP, which recognises astrocytes of radial and stellate morphology in higher and lower vertebrates, should prove a useful agent for studying astrocyte expression during development and regeneration.
1 Dahl, D. and Bignami, A., Immunochemical and immunofluorescence studies of the glial fibrillary acidic protein in vertebrates, Brain Research, 61 (1973) 279-293. 2 Dahl, D., Crosby, C.J., Sethi, J.S. and Bignami, A., Glial fibrillary acidic (GFA) protein in vertebrates: immunofluorescence and immunoblotting study with monoclonal and polyelonal antibodies, J. Comp. Neurol., 239 (1985) 75-88. 3 Hsu, S.M., Raine, L. and Fanger, H., Use of avidin-biotin peroxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabelled antibody (PAP) procedure, J. Histochem. Cytochem,, 29 (1981) 575-580. 4 Liozzi, EJ. and Miller, R.H., Radially oriented astrocytes in the
normal adult rat spinal cord, Brain Research, 403 (1987) 385-388. 5 Nona, S.N., Shehab, S.A.S., Stafford, C.A. and Cronly-Dillon, J.R., Glial fibrillary acidic protein (GFAP) from goldfish: its Iocalisation in visual pathway, Gila, 2 (1989) 189-200. 60ntenienti, B., Kimura, H. and Maeda, T., Comparative studies of the glial fibrillary acidic protein in vertebrates by PAP immunohistochemistry, J. Comp. Neurol., 215 (1983) 427-436. 7 Quitschke, W., Jones, P.S. and Schechter, N., Survey of intermediate filament proteins in optic nerve and spinal cord: evidence for differential expression, J. Neurochem., 44 (1985) 1465-1476. 8 Sternberger, L.A., lmmunocytochemistry, Wiley, Berlin, 1979.
We thank Dr. D. Dahl for the gift of anti-human GFAP, and the Wellcome Trust for financial support.
343
Elsevier
BRES 23847
Anti-goldfish glial fibrillary acidic protein (GFAP) recognises astrocytes from rat CNS Sara S.A. Shehab, Carole A. Stafford, Shmaiel N. Nona and John R. Cronly-Dillon Developmental Neurobiology Laboratory, Department of Optometry and Vision Sciences, U.M. L S. T., Manchester (U. K. )
(Accepted 22 August 1989) Key words: Glial fibrillary acidic protein; Astrocyte; Immunohistochemistry
A polyclonal antibody to goldfish GFAP recognises, immunohistochemically, astrocyte populations in rat brain, spinal cord and optic nerve. The pattern of staining compares favourablywith that obtained using a polyclonalanti-human GFAP or a monoclonal anti-porcine GFAE These results are consistent with the notion that GFAP is well conserved in vertebrate phylogeny. The intermediate filament cytoskeletal fraction from goldfish brain contains a prominent protein, which has the same electrophoretic mobility as the 51 kDa GFAP isolated similarly from rat brain and spinal cord. We have termend this protein goldfish GFAP 5. An antibody produced in rabbit to the purified goldfish GFAP, which cross-reacts with purified rat GFAP on immunobloP, has been used in this study to localise astrocytes in rat CNS. Norwegian black hooded rats, bred in our laboratories, were used in this study. Tissue from cerebrum, cerebellum, spinal cord and optic nerve was quickly frozen in liquid nitrogen and then cut on a cryostat (-20 °C). Sections (20/~m) were picked up onto gelatincoated slides, dried at room temperature for 1 h and then fixed in eithe[ 4% paraformaldehyde in PBS (pH 7.3) or in acetic acid: alcohol (5:95), for 0.5 h at room temperature. Sections were washed with PBS and then incubated with anti-goldfish GFAP 0:200-1:400)or antihuman GFAP (1:400) in PBS at 4 °C in a humid chamber overnight. This was followed by successive hourly treatments, at room temperature, with swine-anti-rabbit (1:50; Dako) and peroxidase-anti-peroxidase a (PAP, 1:100; Dako). Also used was a monoclonal anti-porcine GFAP (1:100-1:200; ICN). The secondary antibody used was biotinylated sheep anti-mouse IgG (1:50; Amersham), and this was followed by streptavidin biotinylated horseradish peroxidase 3 (1:100; Amersbam). The reaction product in both cases was visualised with 3,3"diaminobenzidine/hydrogen peroxide, before being prepared for observation with a Leitz Diaplan light microscope. For double-labelling studies, sections incubated
with primary antibodies were then incubated with either fluorescein (FITC) conjugated goat-anti-mouse IgG (1:50; Sigma), for the monoclonal anti-GFAP, or rhodamine (TRITC) conjugated goat-anti-rabbit IgG (1:50; Sigma), for the polyclonal anti-goldfish GFAP. All three antibodies used in this study recognised astrocytes in rat brain, spinal cord and optic nerve. In cerebeilar white matter, anti-goldfish GFAP recognised astrocytes of stellate morphology (Fig. la). However, while the density of these positive structures was similar to that observed with anti-human GFAP (Fig. lb) and anti-porcine GFAP (not shown), the intensity of the processes was somewhat weaker. In our original report s, it was shown that Bergmann fibres were also clearly stained with anti-goldfish GFAP; here, we further demonstrate, by the use of double labelling, that all the structures recognised by anti-porcine GFAP are also recognised by anti-goldfish GFAP (Figs. 2 and 3). The staining pattern in the hippocampus also revealed astrocytic structures of stellate morphology, with antigoldfish GFAP (Fig. 4a). Here, both the number of positive structures and their intensity were somewhat less than those observed with either anti-porcine GFAP (Fig. 4b), or with anti-human GFAP (not shown). In white matter of spinal cord, stellate as well as radially oriented 4 (Fig. 5), astrocytes were recognised with anti-goldfish GFAP, while in optic nerve GFAP immunoreactivity was in the form of a network, similar to the staining observed with anti-human GFAP (manuscript in preparation). Preahsorption of goldfish antiGFAP with goldfish GFAP showed no positive imn~luno-
Correspondence: S.N. Nona, Developmental Neurology Lab., Dept. Optometry and Vision Sci., U.M.I.S.T., Manchester M60 1QD,U.K.
0006-8993/891503.50© 1989 Elsevier Science Publishers B.V. (Biomedical Division)
Fig. 1. Sagittal sections of rat cerebellar white matter immunolabelled with antibodies to (a) goldfish GFAP and (b) human GFAP (PAP
method). Bar -- 50 ~m.
l'lg. 2. Sagittal sections of rat cerebellum showing Bergmann fibres double-labelled with antibodies to (a) goldfish GFAP (TRITC) and (b) porcine GFAP (FITC). Arrow indicates piai surface. Bar = 100/~m.
Fig. 3. Sagittal sections of rat cerebellar white matter double-labelled with antibodies to (a) goldfish GFAP (TRITC) and (b) porcine GFAP (FITC). Bar -- 100 ~m.
Fig. 4. Coronal sections of brain hippocampal region immunolabelled with antibodies to (a) goldfish GFAP and (b) porcine GFAP (PAP). Bar = 50/~m.
346
Fig. 5. Cross-section of rat spinal cord immunolabelled with anti-goldfish GFAP (PAP) showing the presence of astrocytes of stellate (arrow) and radial (arrowhead) morphology. Bar = 50/zm.
reactivity (56/~g GFAP per mi diluted antibody, incubated at room temperature for 2 h). The above immunohistochemical results confirm that GFAP is well conserved in vertebrate phylogeny. Previous work had shown that antibodies raised against GFAP from human, shark, chicken and bovine cross-react with GFAP from brain and spinal cord of several higher and lower vertebrates, with only a few exceptions t'2'6. One of the main exceptions was goldfish, where anti-human GFAP failed to recognise GFAP-positive glia in rectum 1 or in optic nerve and tract1'7. However, anti-goldfish GFAP described in the present work has been shown in normal goldfish to strongly label ependymal glia in tectum as well as an array of processes in optic tract,
whilst in optic nerve the labelling is very weak s. The fact that this anti-goldfish GFAP also recognises astrocyte populations in rat CNS, as shown in this study, suggests that it has epitopes not only to goldfish GFAP but also to rat GFAP, although these appear to be less abundant in the latter case. The availability of an anti-GFAP, which recognises astrocytes of radial and stellate morphology in higher and lower vertebrates, should prove a useful agent for studying astrocyte expression during development and regeneration.
1 Dahl, D. and Bignami, A., Immunochemical and immunofluorescence studies of the glial fibrillary acidic protein in vertebrates, Brain Research, 61 (1973) 279-293. 2 Dahl, D., Crosby, C.J., Sethi, J.S. and Bignami, A., Glial fibrillary acidic (GFA) protein in vertebrates: immunofluorescence and immunoblotting study with monoclonal and polyelonal antibodies, J. Comp. Neurol., 239 (1985) 75-88. 3 Hsu, S.M., Raine, L. and Fanger, H., Use of avidin-biotin peroxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabelled antibody (PAP) procedure, J. Histochem. Cytochem,, 29 (1981) 575-580. 4 Liozzi, EJ. and Miller, R.H., Radially oriented astrocytes in the
normal adult rat spinal cord, Brain Research, 403 (1987) 385-388. 5 Nona, S.N., Shehab, S.A.S., Stafford, C.A. and Cronly-Dillon, J.R., Glial fibrillary acidic protein (GFAP) from goldfish: its Iocalisation in visual pathway, Gila, 2 (1989) 189-200. 60ntenienti, B., Kimura, H. and Maeda, T., Comparative studies of the glial fibrillary acidic protein in vertebrates by PAP immunohistochemistry, J. Comp. Neurol., 215 (1983) 427-436. 7 Quitschke, W., Jones, P.S. and Schechter, N., Survey of intermediate filament proteins in optic nerve and spinal cord: evidence for differential expression, J. Neurochem., 44 (1985) 1465-1476. 8 Sternberger, L.A., lmmunocytochemistry, Wiley, Berlin, 1979.
We thank Dr. D. Dahl for the gift of anti-human GFAP, and the Wellcome Trust for financial support.