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Miscellaneous Fixatives
6
Miscellaneous Fixatives
RUTHENIUM TETROXIDE Because ruthenium tetroxide ( R u 0 4) is closely related to O s 0 4 , the possibility of its use as a stain in histology (Ranvier, 1887) and as a fixative for plant (Carpenter and Nebel, 1931) and animal tissue (Bahr, 1954) was explored. No further work on its application as a fixative was reported until much later. Membranes in rat kidney and liver (Gaylarde and Sarkany, 1968) and in the ventral lobe of rat prostate (Pelttari and Helminen, 1979) were successfully fixed with this reagent. These membranes appeared thicker than those preserved with other fixatives. Ruthenium tetroxide is a strong oxidizing agent and decomposes readily. It dissolves slowly, and aqueous solutions decompose rapidly even when kept in the dark under cold temperatures. Fresh solutions show a golden yellow color, which after some time turns brownish as a result of the separation of black deposits of the lower oxides of ruthenium. After this change in color the solution is useless as a fixative. Like O s 0 4 , ruthenium tetroxide is superficial in its action, for it penetrates into the tissue very slowly. The claimed advantage of fixation with ruthenium tetroxide is that like the plasma membrane, the nuclear and cytoplasmic membranes appear as triplelayered structures. These triple-layered structure can be clearly seen without additional staining. On the other hand, in many types of tissues fixed with O s 0 4 , although plasma membranes appear triple-layered, most other membranes commonly give the appearance of a single diffuse line. The width of various mem194
195
Dimethylsuberimidate
branes show a remarkable uniformity in cells fixed with ruthenium tetroxide. Connections between the plasma membrane, membranes of the endoplasmic reticulum, and the inner and outer nuclear membranes have been demonstrated in rat kidney and liver tissues fixed with ruthenium tetroxide (Gaylarde and Sarkany, 1968). In contrast to O s 0 4 , ruthenium tetroxide probably reacts strongly with some of the more polar lipids. The fixative also reacts strongly with proteins, glycogen, and monosaccharides (Gaylarde and Sarkany, 1968). The mechanism of interaction of ruthenium tetroxide with various cellular substances is not yet known. Further work is needed to elucidate, for example, the presence of an electronopaque " c o a t i n g " closely apposed to the surface of the plasma membrane in many types of cells fixed with ruthenium. According to a typical procedure, the tissue is prefixed with buffered 4% glutaraldehyde followed by postfixation with buffered 0.1-0.05% ruthenium tetroxide (pH 7.1) for 1 hr at 4°C. Penetration of ruthenium tetroxide into some tissues is poor.
DIMETHYLSUBERIMIDATE Because glutaraldehyde is exceedingly effective in cross-linking the proteins, this dialdehyde is generally unsuitable for immunologic studies because it causes the loss of antigenicity. The dialdehyde also has the disadvantage of being an inhibitor of enzymes and introducing Schiff-positive aldehyde groups into tissue. To circumvent some of these limitations, dimethylsuberimidate (DMS) was introduced as a fixative for light and electron microscopy by Hassell and Hand (1974). McLean and Singer (1970) were the first to use diimidoesters as fixatives for immunoelectron microscopy. The chemical structure of DMS is as follows: +
- C 1 H 2N
II
+
NH2 C1-
II
H 3CO-C-(CH 2) 6-C-OCH 3
Dimethylsuberimidate is a bifunctional reagent that cross-links proteins probably by reacting with a- and e-amino groups. Probably a covalent bond is formed between the carbon adjacent to the amido group in DMS and an amino group in the amino acid, with the release of a mole of alcohol (Hunter and Ludwig, 1972; Wold, 1972). At about pH 7.5, the reaction with α-amino groups dominates, whereas at about pH 9.5, the reaction is primarily with e-amino groups. More extensive cross-linking of proteins is expected at a higher pH because e-amino groups exceed free α-amino groups in proteins. Since lysine is the major source of e-amino groups, this amino acid may react preferentially with DMS (Hartman and Wold, 1967); thus proteins rich in lysine such as histones and collagen may be readily cross-linked.
196
6. M i s c e l l a n e o u s Fixatives +
Since the amid group ( N H 2 ) is located close to the functional groups in the molecule, DMS probably does not alter the net charge of tissue proteins. Even after extensive reaction with certain imidoesters, significant amounts of enzymatic activity and immunologic properties seem to be retained (Hunter and Ludwig, 1972; Wold, 1972). Specimens fixed with DMS seem to show more accurately the aldehyde groups generated by the periodic acid-Schiff technique because DMS is not an aldehyde and does not introduce additional aldehyde groups. It has been indicated that fixation with DMS results in a high retention of enzymatic activity (glucose-6-phosphatase, thiamine pyrophosphatase, and catalase) (Hand and Hassell, 1976). According to Yamamoto and Yasuda (1977), in specimens fixed with DMS, glutamate dehydrogenase retained 50% activity. It has also been demonstrated that DMS retains more glycogen than that observed after fixation with glutaraldehyde. Mitochondrial matrix shows increased electron density compared with that in the glutaraldehyde-fixed specimens (Fig. 6.1). In addition, the nuclei of DMS-fixed tissue specimens are strongly stained with the Feulgen method with little background reaction in the cytoplasm. Microtubules and neurofilaments are also known to preserve better in the presence of DMSO in glutaraldehyde. The DMS fixative solution is prepared by adding the various components in the following order (Hassell and Hand, 1974) (Pierce Chemical Co., Rockford, Illinois): Distilled water NaOH (1 mol/1) Tris base DMS
7.8 ml 1.2 ml 121-182 mg 160-200 mg
Adjust the pH to 9.5 with HCl or NaOH, and 1.0 ml of 0.2 mol/1 CaCl 2 drop by drop. The buffer vehicle has an osmolality of 300 mosmols. Since DMS is unstable in aqueous solutions, the fixative solution should be prepared immediately prior to use. The duration of fixation should not exceed 2 to 3 hr at room temperature. Tris buffer appears to facilitate the penetration of this fixative 2+ into the tissue. The addition of C a reduces the extraction of proteins. The size of the DMS molecule is larger than that of glutaraldehyde, and thus Fig. 6 . 1 . A, hepatocyte from a rat liver fixed with 20 mg of dimethylsuberimidate per ml in 0.15 mol/1 Tris-HCl buffer (pH 9.5) and postfixed with 1% O s 0 4. The nucleus (N) shows marginated chromatin. The rough endoplasmic reticulum (RER) is scattered throughout the cell. Lipid droplets (LD) and glycogen (GLY) are well preserved, but lysosomes (LY) appear pale. Mitochondria (M) appear typical except for a dense matrix. Bile canaliculus (Β). X7760. B, hepatocyte from a rat liver fixed with 2 . 5 % glutaraldehyde in 0.1 mol/1 cacodylate buffer (pH 7.4), and postfixed with 1% O s 0 4. The ultrastructural organization is similar to that in part A, although the nucleoplasm is more granular, mitochondria have a lighter matrix, and glycogen is stained more intensely. X6800. (Hassell and Hand, 1974.)
Dime thylsuberimidate
197
198
6. M i s c e l l a n e o u s Fixatives
its penetration into the tissue is relatively slow. It should be noted that specimens fixed with DMS show swollen Golgi and smooth endoplasmic reticulum, and the quality of ultrastructure preservation is generally less satisfactory than that obtained with glutaraldehyde (Fig. 6.1).
CARBODIIMIDES l-Ethyl-3(3-dimethylaminopropyl)carbodiimide-HCl (WSC) is a watersoluble bifunctional reagent. It has been suggested that it is preferable to glutaraldehyde as a fixative for cytochemical and immunocytological studies. It is thought to cross-link proteins with minimum alteration in their biological activity. It has been indicated that in the specimens fixed with WSC, the enzymatic activity retained is 15% for alcohol dehydrogenase, 70% for glucose-6phosphatase, 7 3 % for ATPase, and 6 1 % for fructose-1,6-diphosphatase (Yamamoto and Yasuda, 1977). These percentages are much higher than those obtained after fixation with aldehydes. Carbodiimide reacts with both carboxyl and amino groups in proteins at neutral pH, forming intermolecular cross-links. It carries a carboxyl group to an adjacent amino group in proteins. The proposed scheme for cross-linking the proteins (Yamamoto and Yasuda, 1977) is as follows:
Protein-C
+
R—N=C—Ν—R'
OH
Η
O Protein-C
N-R C=N-
O Protein-NH 2
ί
s P
// Protein-C
\/
°
Protein
+
II R—N— C— N—R' H
H
Η
At acidic pH values, the protein groups that primarily react with WSC are carboxyl, sulfhydryl, and tyrosine (Carraway and Koshland, 1968; Carraway and
199
Trioxsalen
Triplett, 1970). The optimal pH for protein cross-linking seems to be 7.0 to 7.5, since amino groups in proteins are not very reactive at lower pH values. Carbodiimide was first used for the modification of carboxyl groups in proteins (Scheehan and Hlavka, 1956, 1957). It has been used in immunochemical studies (Goodfriend et al., 1964; Johnson et al., 1966; Linscott et al., 1969) and for preserving tissue fine structure (Yamamoto and Yasuda, 1977). The preparation of the fixative solution (4%) is as follows: WSC Phosphate buffer (0.1 mol/1, pH 7.4)
400 mg 10 ml
The osmalality is 410 mosmols. Specimens are fixed in WSC for 2 hr at 4°C and postfixed with 1% O s 0 4 for 1 hr at 4°C. The WSC solution should be prepared immediately prior to use.
TRIOXSALEN Trioxsalen (4,5',8-trimethylpsoralen) is a trimethyl derivative of psoralen which is a medically important furocoumarin known for its ability to photosensitize mammalian skin (tanning effect). Trioxsalen in conjunction with ultraviolet light of long wavelength (320-380 nm) can covalently cross-link pyrimidines in opposite strands of the DNA double helix (Cole, 1975). The major effect of trioxsalen on DNA migration is assumed to be DNA unwinding. The structure of trioxsalen is as follows: ÇH3
Because trioxsalen permeates plasma and nuclear membranes, it can cross-link DNA in situ in chromatin, in isolated nuclei, or in intact living cells (Pathak and Kramer, 1969). Since DNA within a nucleosome is protected, it is not crosslinked by trioxsalen. In other words, trioxsalen binds at the sites corresponding to the regular nuclease-sensitive regions of the chromatin in nuclei. Consequently, after treatment with trioxsalen, protected regions of DNA appear as a single strand, whereas unprotected regions appear as duplexes due to cross-linking (Lee, 1978). This method is useful in the study of chromatin structure with the TEM, for trioxsalen is able to preserve a linear record of its interaction sites with DNA (Hanson et al., 1976; Wiesehahn et al., 1977; Wiesehahn and Hearst, 1978) and RNA (Wollenzien et al., 1978).
Miscellaneous Fixatives
RUTHENIUM TETROXIDE Because ruthenium tetroxide ( R u 0 4) is closely related to O s 0 4 , the possibility of its use as a stain in histology (Ranvier, 1887) and as a fixative for plant (Carpenter and Nebel, 1931) and animal tissue (Bahr, 1954) was explored. No further work on its application as a fixative was reported until much later. Membranes in rat kidney and liver (Gaylarde and Sarkany, 1968) and in the ventral lobe of rat prostate (Pelttari and Helminen, 1979) were successfully fixed with this reagent. These membranes appeared thicker than those preserved with other fixatives. Ruthenium tetroxide is a strong oxidizing agent and decomposes readily. It dissolves slowly, and aqueous solutions decompose rapidly even when kept in the dark under cold temperatures. Fresh solutions show a golden yellow color, which after some time turns brownish as a result of the separation of black deposits of the lower oxides of ruthenium. After this change in color the solution is useless as a fixative. Like O s 0 4 , ruthenium tetroxide is superficial in its action, for it penetrates into the tissue very slowly. The claimed advantage of fixation with ruthenium tetroxide is that like the plasma membrane, the nuclear and cytoplasmic membranes appear as triplelayered structures. These triple-layered structure can be clearly seen without additional staining. On the other hand, in many types of tissues fixed with O s 0 4 , although plasma membranes appear triple-layered, most other membranes commonly give the appearance of a single diffuse line. The width of various mem194
195
Dimethylsuberimidate
branes show a remarkable uniformity in cells fixed with ruthenium tetroxide. Connections between the plasma membrane, membranes of the endoplasmic reticulum, and the inner and outer nuclear membranes have been demonstrated in rat kidney and liver tissues fixed with ruthenium tetroxide (Gaylarde and Sarkany, 1968). In contrast to O s 0 4 , ruthenium tetroxide probably reacts strongly with some of the more polar lipids. The fixative also reacts strongly with proteins, glycogen, and monosaccharides (Gaylarde and Sarkany, 1968). The mechanism of interaction of ruthenium tetroxide with various cellular substances is not yet known. Further work is needed to elucidate, for example, the presence of an electronopaque " c o a t i n g " closely apposed to the surface of the plasma membrane in many types of cells fixed with ruthenium. According to a typical procedure, the tissue is prefixed with buffered 4% glutaraldehyde followed by postfixation with buffered 0.1-0.05% ruthenium tetroxide (pH 7.1) for 1 hr at 4°C. Penetration of ruthenium tetroxide into some tissues is poor.
DIMETHYLSUBERIMIDATE Because glutaraldehyde is exceedingly effective in cross-linking the proteins, this dialdehyde is generally unsuitable for immunologic studies because it causes the loss of antigenicity. The dialdehyde also has the disadvantage of being an inhibitor of enzymes and introducing Schiff-positive aldehyde groups into tissue. To circumvent some of these limitations, dimethylsuberimidate (DMS) was introduced as a fixative for light and electron microscopy by Hassell and Hand (1974). McLean and Singer (1970) were the first to use diimidoesters as fixatives for immunoelectron microscopy. The chemical structure of DMS is as follows: +
- C 1 H 2N
II
+
NH2 C1-
II
H 3CO-C-(CH 2) 6-C-OCH 3
Dimethylsuberimidate is a bifunctional reagent that cross-links proteins probably by reacting with a- and e-amino groups. Probably a covalent bond is formed between the carbon adjacent to the amido group in DMS and an amino group in the amino acid, with the release of a mole of alcohol (Hunter and Ludwig, 1972; Wold, 1972). At about pH 7.5, the reaction with α-amino groups dominates, whereas at about pH 9.5, the reaction is primarily with e-amino groups. More extensive cross-linking of proteins is expected at a higher pH because e-amino groups exceed free α-amino groups in proteins. Since lysine is the major source of e-amino groups, this amino acid may react preferentially with DMS (Hartman and Wold, 1967); thus proteins rich in lysine such as histones and collagen may be readily cross-linked.
196
6. M i s c e l l a n e o u s Fixatives +
Since the amid group ( N H 2 ) is located close to the functional groups in the molecule, DMS probably does not alter the net charge of tissue proteins. Even after extensive reaction with certain imidoesters, significant amounts of enzymatic activity and immunologic properties seem to be retained (Hunter and Ludwig, 1972; Wold, 1972). Specimens fixed with DMS seem to show more accurately the aldehyde groups generated by the periodic acid-Schiff technique because DMS is not an aldehyde and does not introduce additional aldehyde groups. It has been indicated that fixation with DMS results in a high retention of enzymatic activity (glucose-6-phosphatase, thiamine pyrophosphatase, and catalase) (Hand and Hassell, 1976). According to Yamamoto and Yasuda (1977), in specimens fixed with DMS, glutamate dehydrogenase retained 50% activity. It has also been demonstrated that DMS retains more glycogen than that observed after fixation with glutaraldehyde. Mitochondrial matrix shows increased electron density compared with that in the glutaraldehyde-fixed specimens (Fig. 6.1). In addition, the nuclei of DMS-fixed tissue specimens are strongly stained with the Feulgen method with little background reaction in the cytoplasm. Microtubules and neurofilaments are also known to preserve better in the presence of DMSO in glutaraldehyde. The DMS fixative solution is prepared by adding the various components in the following order (Hassell and Hand, 1974) (Pierce Chemical Co., Rockford, Illinois): Distilled water NaOH (1 mol/1) Tris base DMS
7.8 ml 1.2 ml 121-182 mg 160-200 mg
Adjust the pH to 9.5 with HCl or NaOH, and 1.0 ml of 0.2 mol/1 CaCl 2 drop by drop. The buffer vehicle has an osmolality of 300 mosmols. Since DMS is unstable in aqueous solutions, the fixative solution should be prepared immediately prior to use. The duration of fixation should not exceed 2 to 3 hr at room temperature. Tris buffer appears to facilitate the penetration of this fixative 2+ into the tissue. The addition of C a reduces the extraction of proteins. The size of the DMS molecule is larger than that of glutaraldehyde, and thus Fig. 6 . 1 . A, hepatocyte from a rat liver fixed with 20 mg of dimethylsuberimidate per ml in 0.15 mol/1 Tris-HCl buffer (pH 9.5) and postfixed with 1% O s 0 4. The nucleus (N) shows marginated chromatin. The rough endoplasmic reticulum (RER) is scattered throughout the cell. Lipid droplets (LD) and glycogen (GLY) are well preserved, but lysosomes (LY) appear pale. Mitochondria (M) appear typical except for a dense matrix. Bile canaliculus (Β). X7760. B, hepatocyte from a rat liver fixed with 2 . 5 % glutaraldehyde in 0.1 mol/1 cacodylate buffer (pH 7.4), and postfixed with 1% O s 0 4. The ultrastructural organization is similar to that in part A, although the nucleoplasm is more granular, mitochondria have a lighter matrix, and glycogen is stained more intensely. X6800. (Hassell and Hand, 1974.)
Dime thylsuberimidate
197
198
6. M i s c e l l a n e o u s Fixatives
its penetration into the tissue is relatively slow. It should be noted that specimens fixed with DMS show swollen Golgi and smooth endoplasmic reticulum, and the quality of ultrastructure preservation is generally less satisfactory than that obtained with glutaraldehyde (Fig. 6.1).
CARBODIIMIDES l-Ethyl-3(3-dimethylaminopropyl)carbodiimide-HCl (WSC) is a watersoluble bifunctional reagent. It has been suggested that it is preferable to glutaraldehyde as a fixative for cytochemical and immunocytological studies. It is thought to cross-link proteins with minimum alteration in their biological activity. It has been indicated that in the specimens fixed with WSC, the enzymatic activity retained is 15% for alcohol dehydrogenase, 70% for glucose-6phosphatase, 7 3 % for ATPase, and 6 1 % for fructose-1,6-diphosphatase (Yamamoto and Yasuda, 1977). These percentages are much higher than those obtained after fixation with aldehydes. Carbodiimide reacts with both carboxyl and amino groups in proteins at neutral pH, forming intermolecular cross-links. It carries a carboxyl group to an adjacent amino group in proteins. The proposed scheme for cross-linking the proteins (Yamamoto and Yasuda, 1977) is as follows:
Protein-C
+
R—N=C—Ν—R'
OH
Η
O Protein-C
N-R C=N-
O Protein-NH 2
ί
s P
// Protein-C
\/
°
Protein
+
II R—N— C— N—R' H
H
Η
At acidic pH values, the protein groups that primarily react with WSC are carboxyl, sulfhydryl, and tyrosine (Carraway and Koshland, 1968; Carraway and
199
Trioxsalen
Triplett, 1970). The optimal pH for protein cross-linking seems to be 7.0 to 7.5, since amino groups in proteins are not very reactive at lower pH values. Carbodiimide was first used for the modification of carboxyl groups in proteins (Scheehan and Hlavka, 1956, 1957). It has been used in immunochemical studies (Goodfriend et al., 1964; Johnson et al., 1966; Linscott et al., 1969) and for preserving tissue fine structure (Yamamoto and Yasuda, 1977). The preparation of the fixative solution (4%) is as follows: WSC Phosphate buffer (0.1 mol/1, pH 7.4)
400 mg 10 ml
The osmalality is 410 mosmols. Specimens are fixed in WSC for 2 hr at 4°C and postfixed with 1% O s 0 4 for 1 hr at 4°C. The WSC solution should be prepared immediately prior to use.
TRIOXSALEN Trioxsalen (4,5',8-trimethylpsoralen) is a trimethyl derivative of psoralen which is a medically important furocoumarin known for its ability to photosensitize mammalian skin (tanning effect). Trioxsalen in conjunction with ultraviolet light of long wavelength (320-380 nm) can covalently cross-link pyrimidines in opposite strands of the DNA double helix (Cole, 1975). The major effect of trioxsalen on DNA migration is assumed to be DNA unwinding. The structure of trioxsalen is as follows: ÇH3
Because trioxsalen permeates plasma and nuclear membranes, it can cross-link DNA in situ in chromatin, in isolated nuclei, or in intact living cells (Pathak and Kramer, 1969). Since DNA within a nucleosome is protected, it is not crosslinked by trioxsalen. In other words, trioxsalen binds at the sites corresponding to the regular nuclease-sensitive regions of the chromatin in nuclei. Consequently, after treatment with trioxsalen, protected regions of DNA appear as a single strand, whereas unprotected regions appear as duplexes due to cross-linking (Lee, 1978). This method is useful in the study of chromatin structure with the TEM, for trioxsalen is able to preserve a linear record of its interaction sites with DNA (Hanson et al., 1976; Wiesehahn et al., 1977; Wiesehahn and Hearst, 1978) and RNA (Wollenzien et al., 1978).