- Email: [email protected]
Cytochemical staining methods for the electron microscopic localization of phosphatases and haemoproteins
Micron, 1976, Vol. 7: 287-291. Pergamon Press. Printed in Great Britain.
Cytochemical staining methods for the electron microscopic localization of phosphatases and haemoproteins EDWARD
ESSNER
Kresge Eye Institute, Wayne State University, School of Medicine, Detroit, M I 48201, U.S.A.
Reliable staining methods are currently available for localizing two important classes of enzyme activities; phosphatases and haemoproteins. These activities can be demonstrated by light microscopy using 10pro frozen sections of formol-calcium or glutaraldehyde-fixed tissues and by electron microscopy, using nonfrozen sections prepared on a Smith-Farquhar tissue sectioner (Smith and Farquhar, 1965). Phosphatase activities are useful as markers for cytomembranes and for organelles such as lysosomes and G E R L (see below). Haemoprotein enzymes such as peroxidase and catalase can be demonstrated by using diaminobenzidine (DAB) (Graham and Karnovsky, 1966) as substrate. Methods based on the use of DAB have found wide application and yield localisations of high cytological resolution. Since the intensity, distribution and consistency of enzyme reactions are much easier to judge in frozen sections than in thin sections, localizations should always be assesed by light microscopy prior to electron microscopic study.
reaction product is composed of particles of markedly irregular size and shape (Essner, 1973). When incubations for phosphatase activities are relatively brief, initial deposits of reaction product tend to occur on or adjacent to the face of a particular membrane but, with longer incubations, it may accumulate in the cavities between membranes. Although the deposition of enzyme reaction product on or near membranes may actually reflect the localization of membrane-bound enzymes, such deposits could also be due to diffusion of product (or enzyme) from one site to another. Because of the possibility of diffusion artifact and lack of knowledge concerning the physico-chemical factors involved in the desposition of lead phosphate, such localizations should be interpreted with caution (Essner, 1973). Phosphatase activities have proven useful as 'markers' for cell organelles (Novikoff et al., 1962; Novikoff, 1963; Goldfischer et al., 1964; see also Essner, 1973 for additional references). Acid phosphatase, for example, is a useful marker for lysosomes and for GERL, a specialPhosphatases ized region of Golgi-associated smooth endoThe Gomori-type media (Gomori, 1952) used plasmic reticulum that is involved in the formafor localizing phosphatase activities contain tion of lysosomes (Novikoff et al., 1966, 1971). substrate together with a heavy metal, usually Alkaline phosphatase can serve as a marker for lead, that is used to 'trap' the phosphate plasma membranes in tissues that contain high released during incubation (Essner, 1973). The levels of this enzyme (i.e. brush border of resulting product, lead phosphate, is electron kidney proximal tubule cells and striated border dense and readily visible in thin sections provided of intestinal epithelial cells). Nucleoside phosit accumulates in sufficient amounts in specific phatase activities can be demonstrated in cytosites. For light microscopy, the lead phosphate membranes with various nucleoside, mono, di must be converted to lead sulphide by brief and triphosphates as substrates and manganese exposure to ammonium sulphide. The main as activating ion (Novikoff et al., 1962; Goldadvantages of the lead method over dye pro- fischer et al., 1964). In addition, with the method cedures are; direct (one step) capture of released of Ernst (1972), it appears possible to demonphosphate, easily recognizable reaction product strate, at least in some tissues, a K +-nitrophenyl and applicability to electron microscopy. phosphatase activity ('transport ATP-ase') that Factors that limit cytological resolution are; is thought to reflect K+-dependent dephosfrequent occurrence of 'non-specific' or random phorylation of the Na +, K+-ATP-ase phosprecipitate and the fact that the enzyme phorylated intermediate. 287
CC~ C~
Cytochemical Localization of Phosphatases and Haemoproteins Two phosphatase activities that are useful as markers for the endoplasmic reticulum and nuclear envelope are nucleoside diphosphatase (Novikoff et al., 1962; Goldfischer et al., 1964). (i.e. inosine diphosphate as substrate) and glucose-6 phosphatase (Essner, 1973). The latter enzyme is relatively sensitive to fixation. Nucleoside diphosphatase activity is also demonstrable in the Golgi apparatus (Novikoff and Goldfischer, 1961) (i.e. thiamine pyrophosphate assubstrate) where it is generally restricted to one or two saccules at the inner or 'trans' side of the Golgi stack. Hemoproteins Peroxidase, catalase and other haemoproteins with peroxidatic activity can be visualized for light and electron microscopy using media containing DAB as substrate (Graham and Karnovsky, 1966; Essner, 1974). In frozen sections viewed by light microscopy, oxidized DAB appears as a reddish brown precipitate. Oxidized DAB is also electron dense and easily detected in thin sections at specific sites following the usual period of incubation. Localizations are generally of high cytological resolution. This is due largely to the fact that oxidized DAB precipitates as an amorphous, finely granular deposit. Although diffusion of enzyme reaction product is not commonly observed, it can occur under certain conditions (Novikoff, Novikoff, Q uintana and Davis, 1972). Diffusion of enzyme, apparently caused by prolonged storage of fixed tissue in buffer, has been shown to occur in the case of catalase (Fahimi, 1973). To what extent enzyme diffusion occurs with other haemoproteins is not known. DAB procedures have been used to localize endogenous peroxidase activities in endoplasmic reticulum and nuclear envelope, secretory granules, and certain vesicles and vacuoles in
289
tissues such as thyroid, lacrimal, salivary glands and others (Essner, 1974). These methods have also been useful in identifying the organelles involved in synthesis and packaging of peroxidase, as in the case of maturing granulocytes (Bainton and Farquhar, 1968). The peroxidatic activity of catalase can be visualized in peroxisomes (Novikoffand Goldfischer, 1969). of liver and kidney as well as in microperoxisomes (Novikoff, Novikoff, Davis and Q uintana, 1972) of many tissues. In addition to these applications, the DAB procedures can be used to localize non-enzyme haemoproteins such as haemoglobin and cytochrome c, or exogenous (injected) haemoproteins that are used as tracers in studies of capillary permeability or transepithelial passage of proteins (Essner, 1974). DAB procedures have also been used to localize antibody to specific haemoproteins in immunocompetent cells (Leduc et al., 1969) and as markers for labelling specific antibody (Nakane and Pierce, 1966). More recently, peroxidase and certain other haemoproteins have been used to label concanavalin A where it binds to sites in the cell surface (Bernhard and Avrameas, 1971). Acknowledgements--Supported by NCI grant CA 19648-01 and NIAMD grant 7 RO 1 AM 19386-01.
REFERENCES Bainton, D. F. and Farquhar, M. G., 1965. Differences in enzyme content of azurophit and specific granules of polymorphonuclear leukocytes. II. Cytochemistry and electron microscopy of bone marrow cells, jT. Cell Biol., 39: 299. Bernhard, W. and Avrameas, S., 1971. Ultrastructural visualization of cellular carbohydrate components by means of concanavalin A. Exp. Cell Res., 64: 232. Ernst, S. A., 1972. Transport adenosine triphosphatase cytochemistry. II. Cytochemical localization
Fig. I. Rat transplantable hepatocellular carcinoma; incubated 3min in acid phosphatase medium (cytidine 5'-monophosphate as substrate) (Novikoff, 1963). The reaction product is localized in lysosomes (L) and in a structure (arrow), that probably corresponds to GERL (Novikoff et al., 1966). M, mitochondrion; G, Golgi apparatus, x 23,000. Fig. 2. Rat liver, incubated 10rain in nucleoside diphosphatase Mn 2+ medium (inosine diphosphate as substrate). Initial deposits of reaction product (arrows) are located on or adjacent to membranes of the endoplasmic reticulum and in the space between membranes. M, mitochondrion, x 64,000. Fig. 3. Morris hepatoma (5123 t.c.), incubated for 20see in nucleoside diphosphatase medium (thiamine pyrophosphate as substrate). The reaction product (arrows) is localized in one saccule of each Golgi (G) stack, x 20,000.
29CI
E d w a r d Essncr
Cytochemical Localization of Phosphatases and Haemoproteins of ouabain-sensitive, potassium-dependent phosphatase activity in the secretory epithelium of the avian salt gland. J. Histochem. Cytochem. 20: 23. Essner, E., 1973. Phosphatases. In: Electron Microscopy of Enzymes: Principles and Methods, Vol. 1 (Hayat, M. A. (ed.), pp. 44-76, Van Nostrand-Reinhold, New York. Essner, E., 1974. Haemoproteins. In: Electron Microscopy of Enzymes: Principles and Methods, Vol. 2, p. 1 (Hayat, M. A., ed.). Van Nostrand-Reinhold, New York. Fahimi, H. D., 1973. Diffusion artifacts in cytochemistry of catalase. J. Histochem. Cytochem., 21: 999. Goldfischer, S., Essner, E. and Novikoff, A. B., 1964. The localization of phosphatase activities at the level of ultrastructure. J. Histochem. Cytochem., 12: 72. Gomori, G. 1952. Microscopic Histochemistry : Principles and Practice. Chicago, Univ. of Chicago Press. Graham, R. C. and Karnovsky, M. J., 1966. The early stages of absorption of injected horseradish peroxidase in the proximal tubules of mouse kidney. Ultrastructural cytochemistry by a new technique. J. Histochem. Cytochem., 14: 291. Leduc, E. H., Scott, G. B. and Avrameas, S., 1969. Ultrastructural localization ofintracellular immune globulins in plasma cell and lymphoblast by enzyme-labeled antibodies. J. Histochem. Cytochem., 17:211. Nakane, P. K. and Pierce, G. B., 1966. Enzymelabeled antibodies: Preparation and application for localization of antigens. J. Histochem. Cytochem., 14: 929. Novikoff, A. B. and Goldfischer, S., 1961. Nucleoside-
291
phosphatase activity in the Golgi apparatus and its usefulness for cytological studies. Proc. Nat. Acad. Sci. U.S.A., 47" 803. Novikoff, A. B., Essner, E., Goldfischer, S. and Heus, M., 1962. Nucleoside phosphatase activities of cytomembranes. Symp° Intern. Soc. Cell Biol., 1: 149. Novikoff, A. B., 1963. Lysosomes in the physiology and pathology of cells: contributions of staining methods. In : Ciba Foundation Symposium on Lysosomes, de Reuck, A. V. S. and Cameron, M. P. (eds.), Little, Brown & Co., Boston, 36. Novikoff, A. B., Roheim, P. S. and Quintana, N., 1966. Changes in rat liver cells induced by orotic acid feeding. Lab. Invest., 15: 27. Novikoff, A. B. and Goldfischer, S., 1969. Visualization of peroxisomes (microbodies) and mitochondria with diaminobenzidine. J. Histochem. Cytochem., 17: 675. Novikoff, P. M., Novikoff, A. B., Quintana, N., and Hauw, J. J., 1971. Golgi apparatus, G E R L and lysosomes of neurones in rat dorsal root ganglia: Studies by thick and thin section cytochemistry. J. Cell Biol. 50: 859. Novikoff, A. B., Novikoff, P. M., Davis, C. and Quintana, N., 1972. Studies on microperoxisomes. II. A cytochemical method for light and electron microscopy. J. Histochem. Cytochem., 20: 1006. Novikoff, A. B., Novikoff, P. M., Q uintana, N. and Davis, C., 1972. Diffusion artifacts in 3, 3'-diaminobenzidine cytochemistry. J. Histoehem. @tochem., 20: 745. Smith, R. E. and Farquhar, M. G., 1975. Preparation of nonfrozen sections for electron microscope cytochemistry. Scient. Instrum. News RCA, 10: 13.
Fig. 4. Rat exorbital lacrimal gland, incubated 4hr in DAB medium, pH 8.0. Oxidized DAB is localized in cisternae of rough endoplasmic reticulum and in the nuclear envelope (arrows). N, nucleus. × 12,000. Fig. 5. Rat intraorbital lacrimal gland, incubated 60min in DAB medium. Oxidized DAB is deposited in cisternae of rough endoplasmic reticulum (arrow). There is no indication of diffusion of reaction product. M, mitochondrion. X 64,000. Figs. 6. Rat liver incubated 60 rain in DAB medium, p H 9.0. The reaction product is localized in peroxisomes; none is seen in mitochondria (M) or other structures. The arrow points to nucleoid of peroxisome, x 40,000. Fig. 7. Mouse liver, 2min after intravenous injection of 10rag horseradish peroxidase. Incubated for 10min in DAB medium. The reaction product is localized in channels (arrows) and in pinocytotic vesicles at the sinusoidal (lower portion of micrograph) surface of the hepatocyte. x 40,000.
Cytochemical staining methods for the electron microscopic localization of phosphatases and haemoproteins EDWARD
ESSNER
Kresge Eye Institute, Wayne State University, School of Medicine, Detroit, M I 48201, U.S.A.
Reliable staining methods are currently available for localizing two important classes of enzyme activities; phosphatases and haemoproteins. These activities can be demonstrated by light microscopy using 10pro frozen sections of formol-calcium or glutaraldehyde-fixed tissues and by electron microscopy, using nonfrozen sections prepared on a Smith-Farquhar tissue sectioner (Smith and Farquhar, 1965). Phosphatase activities are useful as markers for cytomembranes and for organelles such as lysosomes and G E R L (see below). Haemoprotein enzymes such as peroxidase and catalase can be demonstrated by using diaminobenzidine (DAB) (Graham and Karnovsky, 1966) as substrate. Methods based on the use of DAB have found wide application and yield localisations of high cytological resolution. Since the intensity, distribution and consistency of enzyme reactions are much easier to judge in frozen sections than in thin sections, localizations should always be assesed by light microscopy prior to electron microscopic study.
reaction product is composed of particles of markedly irregular size and shape (Essner, 1973). When incubations for phosphatase activities are relatively brief, initial deposits of reaction product tend to occur on or adjacent to the face of a particular membrane but, with longer incubations, it may accumulate in the cavities between membranes. Although the deposition of enzyme reaction product on or near membranes may actually reflect the localization of membrane-bound enzymes, such deposits could also be due to diffusion of product (or enzyme) from one site to another. Because of the possibility of diffusion artifact and lack of knowledge concerning the physico-chemical factors involved in the desposition of lead phosphate, such localizations should be interpreted with caution (Essner, 1973). Phosphatase activities have proven useful as 'markers' for cell organelles (Novikoff et al., 1962; Novikoff, 1963; Goldfischer et al., 1964; see also Essner, 1973 for additional references). Acid phosphatase, for example, is a useful marker for lysosomes and for GERL, a specialPhosphatases ized region of Golgi-associated smooth endoThe Gomori-type media (Gomori, 1952) used plasmic reticulum that is involved in the formafor localizing phosphatase activities contain tion of lysosomes (Novikoff et al., 1966, 1971). substrate together with a heavy metal, usually Alkaline phosphatase can serve as a marker for lead, that is used to 'trap' the phosphate plasma membranes in tissues that contain high released during incubation (Essner, 1973). The levels of this enzyme (i.e. brush border of resulting product, lead phosphate, is electron kidney proximal tubule cells and striated border dense and readily visible in thin sections provided of intestinal epithelial cells). Nucleoside phosit accumulates in sufficient amounts in specific phatase activities can be demonstrated in cytosites. For light microscopy, the lead phosphate membranes with various nucleoside, mono, di must be converted to lead sulphide by brief and triphosphates as substrates and manganese exposure to ammonium sulphide. The main as activating ion (Novikoff et al., 1962; Goldadvantages of the lead method over dye pro- fischer et al., 1964). In addition, with the method cedures are; direct (one step) capture of released of Ernst (1972), it appears possible to demonphosphate, easily recognizable reaction product strate, at least in some tissues, a K +-nitrophenyl and applicability to electron microscopy. phosphatase activity ('transport ATP-ase') that Factors that limit cytological resolution are; is thought to reflect K+-dependent dephosfrequent occurrence of 'non-specific' or random phorylation of the Na +, K+-ATP-ase phosprecipitate and the fact that the enzyme phorylated intermediate. 287
CC~ C~
Cytochemical Localization of Phosphatases and Haemoproteins Two phosphatase activities that are useful as markers for the endoplasmic reticulum and nuclear envelope are nucleoside diphosphatase (Novikoff et al., 1962; Goldfischer et al., 1964). (i.e. inosine diphosphate as substrate) and glucose-6 phosphatase (Essner, 1973). The latter enzyme is relatively sensitive to fixation. Nucleoside diphosphatase activity is also demonstrable in the Golgi apparatus (Novikoff and Goldfischer, 1961) (i.e. thiamine pyrophosphate assubstrate) where it is generally restricted to one or two saccules at the inner or 'trans' side of the Golgi stack. Hemoproteins Peroxidase, catalase and other haemoproteins with peroxidatic activity can be visualized for light and electron microscopy using media containing DAB as substrate (Graham and Karnovsky, 1966; Essner, 1974). In frozen sections viewed by light microscopy, oxidized DAB appears as a reddish brown precipitate. Oxidized DAB is also electron dense and easily detected in thin sections at specific sites following the usual period of incubation. Localizations are generally of high cytological resolution. This is due largely to the fact that oxidized DAB precipitates as an amorphous, finely granular deposit. Although diffusion of enzyme reaction product is not commonly observed, it can occur under certain conditions (Novikoff, Novikoff, Q uintana and Davis, 1972). Diffusion of enzyme, apparently caused by prolonged storage of fixed tissue in buffer, has been shown to occur in the case of catalase (Fahimi, 1973). To what extent enzyme diffusion occurs with other haemoproteins is not known. DAB procedures have been used to localize endogenous peroxidase activities in endoplasmic reticulum and nuclear envelope, secretory granules, and certain vesicles and vacuoles in
289
tissues such as thyroid, lacrimal, salivary glands and others (Essner, 1974). These methods have also been useful in identifying the organelles involved in synthesis and packaging of peroxidase, as in the case of maturing granulocytes (Bainton and Farquhar, 1968). The peroxidatic activity of catalase can be visualized in peroxisomes (Novikoffand Goldfischer, 1969). of liver and kidney as well as in microperoxisomes (Novikoff, Novikoff, Davis and Q uintana, 1972) of many tissues. In addition to these applications, the DAB procedures can be used to localize non-enzyme haemoproteins such as haemoglobin and cytochrome c, or exogenous (injected) haemoproteins that are used as tracers in studies of capillary permeability or transepithelial passage of proteins (Essner, 1974). DAB procedures have also been used to localize antibody to specific haemoproteins in immunocompetent cells (Leduc et al., 1969) and as markers for labelling specific antibody (Nakane and Pierce, 1966). More recently, peroxidase and certain other haemoproteins have been used to label concanavalin A where it binds to sites in the cell surface (Bernhard and Avrameas, 1971). Acknowledgements--Supported by NCI grant CA 19648-01 and NIAMD grant 7 RO 1 AM 19386-01.
REFERENCES Bainton, D. F. and Farquhar, M. G., 1965. Differences in enzyme content of azurophit and specific granules of polymorphonuclear leukocytes. II. Cytochemistry and electron microscopy of bone marrow cells, jT. Cell Biol., 39: 299. Bernhard, W. and Avrameas, S., 1971. Ultrastructural visualization of cellular carbohydrate components by means of concanavalin A. Exp. Cell Res., 64: 232. Ernst, S. A., 1972. Transport adenosine triphosphatase cytochemistry. II. Cytochemical localization
Fig. I. Rat transplantable hepatocellular carcinoma; incubated 3min in acid phosphatase medium (cytidine 5'-monophosphate as substrate) (Novikoff, 1963). The reaction product is localized in lysosomes (L) and in a structure (arrow), that probably corresponds to GERL (Novikoff et al., 1966). M, mitochondrion; G, Golgi apparatus, x 23,000. Fig. 2. Rat liver, incubated 10rain in nucleoside diphosphatase Mn 2+ medium (inosine diphosphate as substrate). Initial deposits of reaction product (arrows) are located on or adjacent to membranes of the endoplasmic reticulum and in the space between membranes. M, mitochondrion, x 64,000. Fig. 3. Morris hepatoma (5123 t.c.), incubated for 20see in nucleoside diphosphatase medium (thiamine pyrophosphate as substrate). The reaction product (arrows) is localized in one saccule of each Golgi (G) stack, x 20,000.
29CI
E d w a r d Essncr
Cytochemical Localization of Phosphatases and Haemoproteins of ouabain-sensitive, potassium-dependent phosphatase activity in the secretory epithelium of the avian salt gland. J. Histochem. Cytochem. 20: 23. Essner, E., 1973. Phosphatases. In: Electron Microscopy of Enzymes: Principles and Methods, Vol. 1 (Hayat, M. A. (ed.), pp. 44-76, Van Nostrand-Reinhold, New York. Essner, E., 1974. Haemoproteins. In: Electron Microscopy of Enzymes: Principles and Methods, Vol. 2, p. 1 (Hayat, M. A., ed.). Van Nostrand-Reinhold, New York. Fahimi, H. D., 1973. Diffusion artifacts in cytochemistry of catalase. J. Histochem. Cytochem., 21: 999. Goldfischer, S., Essner, E. and Novikoff, A. B., 1964. The localization of phosphatase activities at the level of ultrastructure. J. Histochem. Cytochem., 12: 72. Gomori, G. 1952. Microscopic Histochemistry : Principles and Practice. Chicago, Univ. of Chicago Press. Graham, R. C. and Karnovsky, M. J., 1966. The early stages of absorption of injected horseradish peroxidase in the proximal tubules of mouse kidney. Ultrastructural cytochemistry by a new technique. J. Histochem. Cytochem., 14: 291. Leduc, E. H., Scott, G. B. and Avrameas, S., 1969. Ultrastructural localization ofintracellular immune globulins in plasma cell and lymphoblast by enzyme-labeled antibodies. J. Histochem. Cytochem., 17:211. Nakane, P. K. and Pierce, G. B., 1966. Enzymelabeled antibodies: Preparation and application for localization of antigens. J. Histochem. Cytochem., 14: 929. Novikoff, A. B. and Goldfischer, S., 1961. Nucleoside-
291
phosphatase activity in the Golgi apparatus and its usefulness for cytological studies. Proc. Nat. Acad. Sci. U.S.A., 47" 803. Novikoff, A. B., Essner, E., Goldfischer, S. and Heus, M., 1962. Nucleoside phosphatase activities of cytomembranes. Symp° Intern. Soc. Cell Biol., 1: 149. Novikoff, A. B., 1963. Lysosomes in the physiology and pathology of cells: contributions of staining methods. In : Ciba Foundation Symposium on Lysosomes, de Reuck, A. V. S. and Cameron, M. P. (eds.), Little, Brown & Co., Boston, 36. Novikoff, A. B., Roheim, P. S. and Quintana, N., 1966. Changes in rat liver cells induced by orotic acid feeding. Lab. Invest., 15: 27. Novikoff, A. B. and Goldfischer, S., 1969. Visualization of peroxisomes (microbodies) and mitochondria with diaminobenzidine. J. Histochem. Cytochem., 17: 675. Novikoff, P. M., Novikoff, A. B., Quintana, N., and Hauw, J. J., 1971. Golgi apparatus, G E R L and lysosomes of neurones in rat dorsal root ganglia: Studies by thick and thin section cytochemistry. J. Cell Biol. 50: 859. Novikoff, A. B., Novikoff, P. M., Davis, C. and Quintana, N., 1972. Studies on microperoxisomes. II. A cytochemical method for light and electron microscopy. J. Histochem. Cytochem., 20: 1006. Novikoff, A. B., Novikoff, P. M., Q uintana, N. and Davis, C., 1972. Diffusion artifacts in 3, 3'-diaminobenzidine cytochemistry. J. Histoehem. @tochem., 20: 745. Smith, R. E. and Farquhar, M. G., 1975. Preparation of nonfrozen sections for electron microscope cytochemistry. Scient. Instrum. News RCA, 10: 13.
Fig. 4. Rat exorbital lacrimal gland, incubated 4hr in DAB medium, pH 8.0. Oxidized DAB is localized in cisternae of rough endoplasmic reticulum and in the nuclear envelope (arrows). N, nucleus. × 12,000. Fig. 5. Rat intraorbital lacrimal gland, incubated 60min in DAB medium. Oxidized DAB is deposited in cisternae of rough endoplasmic reticulum (arrow). There is no indication of diffusion of reaction product. M, mitochondrion. X 64,000. Figs. 6. Rat liver incubated 60 rain in DAB medium, p H 9.0. The reaction product is localized in peroxisomes; none is seen in mitochondria (M) or other structures. The arrow points to nucleoid of peroxisome, x 40,000. Fig. 7. Mouse liver, 2min after intravenous injection of 10rag horseradish peroxidase. Incubated for 10min in DAB medium. The reaction product is localized in channels (arrows) and in pinocytotic vesicles at the sinusoidal (lower portion of micrograph) surface of the hepatocyte. x 40,000.