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The substrate film method in enzyme histochemistry
Experimental
40
Cell Research, Suppl. 7, 40-19 (1959)
THE SUBSTRATE FILM METHOD IN ENZYME HISTOCHEMISTRYl R. DAOUSTe Research Laboratories, Montreal Cancer Institute, Notre Dame Hospital University of Montreal, Montreal, Canada
and
THE present
paper will describe the studies carried out during the past few years on the localization of enzyme activities in tissue sections by the substrate film method. Essentially, the method is to place tissue sections in contact with a film composed of gelatine and a substrate, to allow the tissue enzyme to act upon its substrate in the film and, after exposure, to stain the unattacked substrate remaining in the film. The substrate is transformed in the parts of the film covering the areas of tissue sections containing the appropriate enzyme and retained in the parts overlying the inactive regions. Thus, a reaction pattern is observed in the film after staining, and comparison of this “autograph” with the corresponding tissue sections reveals the sites of enzyme activity in the latter. The studies so far carried out on the substrate film method may be divided into three parts: (a) first, a method was devised for localizing deoxyribonuclease (DNAase) activity in tissue sections; (b) the method for DNAase was then used for investigating a particular problem, namely the distribution of DNAase in normal, cirrhotic and neoplastic rat livers; (c) more recently, the substrate film method was adapted to the localization of a second enzyme, ribonuclease (RNAase), in tissue sections. The results of these investigations will be described here together with a general view of the possible applications of the substrate film method.
LOCALIZATION
OF DNAase
IN
TISSUE
SECTIONS
The first enzyme localized by the use of films of substrate was the enzyme DNAase which hydrolyzes deoxyribonucleic acid (DNA) [2]. Films of gelatine and DNA were prepared on glass slides, fixed in formaldehyde to render the DNA insoluble in water, washed in water and allowed to dry. The tissue 1 Investigations supported by the National Cancer Institute of Canada. $ Research Associate of the National Cancer Institute of Canada. Experimental
Cell Research, Suppl. 7
Substrate film method
41
sections, prepared from fresh frozen tissues, were mounted on slides covered films were placed in conwith gelatine-glycerol (Fig. 1 a). The gelatine-DNA tact with the tissue sections for different lengths of time (5 to 60 minutes) at room temperature (Fig. 1 b). The films were then separated from the tissue sections, washed in water and the residual DNA stained with toluidine blue. The tissue sections were fixed in formaldehyde, washed in water and also stained with toluidine blue. Unstained areas were observed in the parts of the films exposed to tissue sections (Fig. 1 c).
GELATINE - GLYCEROL GLASS SLIDE
GLASS SLIDE ~~sA$N~E-C~~~NFILM
(23
GELATINE -GLYCEROL GLASS SUDE
Fig. l.-Localization of DNAase in tissue sections by the substrate fiim method; (a) materials used in their relative positions before exposing the DNA film to tissue sections, (b) cross section of the same elements during exposure and (c) DNA film and tissue sections after separation and staining with toluidine blue.
@
~~~s!i!gE,~N
Fig. 2.-DNA films exposed to control and DNAase solutions and stained with toluidine blue. Experimental
Cell Research, Suppl. 7
Fig. 3.-DNA
film exposed to sections of small intestine
shown in Fig. 4. x 2.
The control studies carried out on DNA films may be summarized as follows. The fixed gelatine-DNA films remained intact in water and control solutions while a progressive loss of DNA was observed in films standing in DNAase solution (Fig. 2). Similar films exposed to blood serum became progressively toluidine-blue negative thus duplicating the results obtained with the DNAase solution. Exposure of films to tissue sections resulted in a loss of DNA in those regions in contact with tissue sections (Figs. 3 and 4) while no effect was observed in films exposed to inactivated tissues (90°C for 10 minutes). So far as the supporting medium is concerned, no effect was observed in control gelatine films placed in contact with tissue sections and stained with phloxine. It was concluded from these tests that the removal of DNA in films exposed to tissue sections was due to the hydrolytic action of tissue DNAase and the method appeared reliable for studying the distribution of this enzyme in tissue sections. In the small intestine of the rat (Figs. 5 and 6), DNAase activity was found mainly in the lumen between the villi as shown by the white (toluidine-blue negative) pattern left in the film. The epithelial cells of the villi and the crypts of Lieberktihn were also active. Presumably, DNAase is contained in the goblet cells of the villi and crypt epithelia and is discharged into the lumen together with the mucus. Weak reactions were given by the lamina propria, the submucosa and the muscle layers. In films exposed to sections of the pancreas, a reaction pattern corresponding to the acinar cells was obtained. This suggests that the pancreatic DNAase which reacts with labeled antibodies [7]. or part at least of the protein, represents the active form of the enzyme. In the thyroid (Figs. 7 and 8), DNAase activity was found mainly in the colloid, the follicular cells themselves being relatively inactive. Positive reactions were also given by the interfolicular tissue. Experimental
Cell Research, Suppl. 7
Substrate film method
Fig. B.-DNA Fig. ‘I.-DNA
43
film exposed to section of small intestine shown in Fig. 6. x 40. film exposed to section of the thyroid shown in Fig. 8. x 40.
DISTRIBUTION IN
RAT
LIVER
DURING
OF DNAaee CARCINOGENESIS
Following the examination of DNAase distribution in a few normal tissues, the DNA film method was applied to the investigation of a particular problem, namely the distribution of DNAase in rat liver during carcinogenesis by the azo-dye 4-dimethylaminoazobenzene, or DAB [5]. The DNAase activity of normal, cirrhotic and neoplastic livers had been determined previously in our laboratory by biochemical assays on tissue homogenates and slight differences only had been observed between the enzymatic activities of these tissues [5, 61. The biochemical data, however, represent mean activities of several cell types and different extracellular fluids and do not give any insight into the changes which may take place in particular tissue elements [l, 3, 41. Thus, to obtain information on the DNAase activity of the liver structural Experimental
Cell Research, Suppl. 7
R. Daousf units during carcinogenesis, the problem was investigated by histochemicar analysis. DNA films exposed to normal liver (Figs. 9 and 10) showed a positive reacSlight DNAase activity was also tion corresponding to liver parenchyma. observed in the blood vessels indicated by arrows in the same figures. In cirrhotic liver, intense DNAase activity was found in the hyperplastic parenchymal nodules (Figs. 11 and 12). IThe trabeculae composed of bile ducts and connective tissue elements were relatively inactive. The multifocal reaction observed in the trabeculae probably corresponds to groups of parenchymal cells scattered among the bile ducts and connective tissue. A peculiar feature observed in cirrhotic liver was the fact that some hyperplastic nodules were partly positive and partly negative (Figs. 13 and 14). It appeared especially interesting to note that such inactive regions of cirrhotic livers were composed of parenchymal cells which, on the basis of their architectural arrangement and basophilic properties, are often considered as the sites of origin of liver tumors. The fiber tumors (hepatomas), for their part, showed negligible DNAase activity compared with the surrounding non-tumoral tissue (Figs. 15 and 16). The weak reaction observed in the tumor.mass corresponds to trabeculae of bile duct cells and connective tissue. A further example of the low DNAase activity of the tumor cells is presented in Figs. 17 and 18 (lower part). The necrotic regions of the tumors, on the other hand, were highly active (upper parts and bottom left, corners of the same figures). The most interesting finding in this study was the fact that groups of parenchymal cells lost their DNAase activity in cirrhotic liver and that the tumor cells were practically inactive. This problem is being ‘further investigated. Attempts are also being made in this laboratory to determine whether the loss of DNAase activity by liver cells is an important factor in-tumor formation. LOCALIZATION
OF
RNAase
ACTIVITY
IN’
TISSUE
SECTIONS
During the past year, in collaboration with Mrs. H. Amano, attempts have been made to adapt the substrate film method to the localization of a second enzyme, RNAase, which hydrolyses ribonucleic acid (RNA). The procedure used at first for localizing RNAase was exactly the same as Fia. Fig. Fig. Fig. Fig.
9.-DNA film exuosed to section of normal liver shown in Fig. 10. d30. ll.-DNA film exposed to section of cirrhotic liver shown in kg. 12. x 30. 13.-DNA film exposed to section of cirrhotic liver shown in Fig. 14. x 30. 15.-DNA film exposed to section of liver tumor (hepatoma) shiwn in Fig. 16. x 30. 17.-DNA film exposed to section of necrotic liver tumor (hepatoma) shown in Fig. 18. x 30.
Experimental
Cell Research, Suppl. 7
Substrate film method
Experimental
45
Cell Research, Suppl. 7
R. Daoust that followed for DNAase except that RNA was utilized in the film instead of DNA. The fixed gelatine-RNA films exposed to control solutions were found to remain intact while a progressive loss of RNA resulted from treatment by a RNAase solution (Fig. 19). The other control studies carried out with these films gave the same results as those obtained with the DNA films. The RNA films exposed to tissue sections revealed the sites of RNAase activity in the latter and the resolution was comparable with that obtained for DNAase. In order to increase the resolution of the method, however, a different technique of film preparation was tried. Instead of preparing the films by simply spreading one drop of the gelatine-substrate mixture on the glass slide (spreading technique), 3-4 drops of the mixture were rapidly spread on the slide and the excess allowed to drain by holding the slide vertically on a piece of filter paper (draining technique). The films prepared in this way were much thinner than those obtained by the former technique and, as expected, permitted the localization of RNAase in tissue sections with a much higher resolution. The RNAase distributions reported below are examples of the results obtained with the thinner films. In the small intestine of the rat (Fig. 20) RNAase activity was especially concentrated in the lamina propria and the submucosa. Appreciable reactions were also given by the epithelial cells of the villi and the crypts of Lieberktihn. The muscle layers, on the other hand, were relatively inactive. The distribution of RNAase in the large intestine (Fig.121) was similar to that observed in the small intestine. High RNAase activities were observed in the lamina propria and the submucosa and the epithelial cells were also active. The muscularis mucosae was relatively inactive as well as the muscle layers. Fig. 21 shows an additional structure, a lymphatic nodule, which is negative. In the liver (Fig. 22), a general reaction was observed corresponding to the parenchyma while slight RNAase activity was found in the blood vessels. In the pancreas (Fig. 23), the acinar cells gave a positive reaction and some activity was also present in the intercellular spaces. The Islets of Langerhans (arrows) were relatively inactive. The Islets of Langerhans do not react either with RNAase antibodies (J. M. Marshall, personal communication) indicating that RNAase is practically absent (rather than inhibited) in these locations. POSSIBLE
APPLICATIONS
OF THE
SUBSTRATE
FILM
METHOD
The film methods for DNAase and RNAase can, in their present state, be applied to problems of several types and there is no doubt that future improvements of the method will widen the scope of the present approach. Experimental
Cell Research, Suppl. 7
Substrate jilm method
Fig. Fig. Fig. Fig.
20.-RNA 21.--RNA 22.-RNA 23.-RNA
film film film film
exposed exposed exposed exposed
to to t; to
section section section section
of of of of
47
small intestine. x 60. large intestine. x 60. liver. x 30. pancreas. x 30. Experimental
Cell Research, Suppl. 7
48
R. Daousf
Before concluding, let us esamine method seems to offer: Localization
of several different
briefly
the possibilities
the substrate
film
enzymes in tissue sections
It is probable that suitable films of a variety of substrates can be prepared and, provided that the products of the reactions can be distinguished from the substrates by staining or other procedures, such films could be applied to the localization of many different enzymes in tissues. Distribution
of enzyme activity
in fresh and fixed tissues
The films of substrate being applicable to fresh or fixed tissue sections, the method would appear especially convenient for examining the effect of fixation and other histological procedures on the activity and distribution of enzymes in tissue sections. Comparison
of enzymes in pure solutions,
biological
fluids and tissue sections
The films of substrate may be exposed to enzyme solutions, biological fluids and tissue sections, and may be useful for the comparison of the properties of tissue enzymes with those of purified enzymes. Enzyme activity
of isolated cells, nuclei and mitochondria
Films of substrate could be placed in contact with smears of isolated cells, nuclei or cell particles for studying the intracellular distribution of enzymes; the results obtained with such smears might also be compared with those obtained with sections of whole cells, nuclei or cell particles suspended in gelatine for studying the diffusion of enzymes through cell, nuclear and mitochondrial membranes. Coupling
of the substrate film method with the fluorescent antibody
method
The substrate film method localizes the active enzymes of tissues while the fluorescent antibody reaction localizes the enzyme proteins; comparing the results obtained by. these different methods might permit localization of inactive enzymes in tissue sections. Coupling
of the substrate film method with the precipitation
method
The substrate film method presumably localizes the enzymes acting at the pH of the cells while the precipitation method may reveal the distribution of enzymes at various pH values and in the presence of inhibitors or activaExperimental
Cell Research, Suppl. 7
Substrate film method
49
tom. Comparing the results obtained with such different methods might allow the localization of inhibited or activated enzymes in tissue sections. Thus, the substrate film method seems to offer many interesting possibilities for histochemical studies on enzymes. Efforts, at the moment, are still mainly concentrated on improving the method but it is also hoped to investigate soon some of its possible applications. Evidently, it is only when more results will have been obtained that one will be able to evaluate the scope of this present approach. SUMMARY Studies on the localization of enzyme activities in tissue sections by the substrate film method are described. Results are presented on the distribution of deoxyribonuclease and ribonuclease activities in sections of normal and pathological tissues and possible applications of the general method were discussed. I wish to thank Dr. A. Cantero, Director of .the Research Laboratories, Montreal Cancer Institute, for his continued interest in the present studies. Part of the work described in this paper was carried out at the Chester Beatty Research Institute in London and I am very grateful to Professor A. Haddow for his cooperation and interest. REFERENCES 1. DAOUST, R., J. Natl. Cancer Inst. 15, 1447 (1955). 2. Expfl. Cell Research 12, 203 (1957). 3. in Brauer, R., ed., Liver Function, p. 3. Am. Inst. Biol. SC., Washington, 1958: 4. Acta Unio. Intern. Contra Cancrum, in press. 5. DAOUST, R. and CANTERO, A., J. Histochkm. ‘Cytochem. 7, 139 (1959). 6. LAMIRANDE,G. DE, ALLARD, C. and CANTERO, A., Canad. J. Biochem. Physiol. 32, 35 (1954). 7. MARSHALL, J. M., Exptl. Cell Research 6 240 (1954).
4- 593710
Experimental
Cell Research, Suppl. 7
40
Cell Research, Suppl. 7, 40-19 (1959)
THE SUBSTRATE FILM METHOD IN ENZYME HISTOCHEMISTRYl R. DAOUSTe Research Laboratories, Montreal Cancer Institute, Notre Dame Hospital University of Montreal, Montreal, Canada
and
THE present
paper will describe the studies carried out during the past few years on the localization of enzyme activities in tissue sections by the substrate film method. Essentially, the method is to place tissue sections in contact with a film composed of gelatine and a substrate, to allow the tissue enzyme to act upon its substrate in the film and, after exposure, to stain the unattacked substrate remaining in the film. The substrate is transformed in the parts of the film covering the areas of tissue sections containing the appropriate enzyme and retained in the parts overlying the inactive regions. Thus, a reaction pattern is observed in the film after staining, and comparison of this “autograph” with the corresponding tissue sections reveals the sites of enzyme activity in the latter. The studies so far carried out on the substrate film method may be divided into three parts: (a) first, a method was devised for localizing deoxyribonuclease (DNAase) activity in tissue sections; (b) the method for DNAase was then used for investigating a particular problem, namely the distribution of DNAase in normal, cirrhotic and neoplastic rat livers; (c) more recently, the substrate film method was adapted to the localization of a second enzyme, ribonuclease (RNAase), in tissue sections. The results of these investigations will be described here together with a general view of the possible applications of the substrate film method.
LOCALIZATION
OF DNAase
IN
TISSUE
SECTIONS
The first enzyme localized by the use of films of substrate was the enzyme DNAase which hydrolyzes deoxyribonucleic acid (DNA) [2]. Films of gelatine and DNA were prepared on glass slides, fixed in formaldehyde to render the DNA insoluble in water, washed in water and allowed to dry. The tissue 1 Investigations supported by the National Cancer Institute of Canada. $ Research Associate of the National Cancer Institute of Canada. Experimental
Cell Research, Suppl. 7
Substrate film method
41
sections, prepared from fresh frozen tissues, were mounted on slides covered films were placed in conwith gelatine-glycerol (Fig. 1 a). The gelatine-DNA tact with the tissue sections for different lengths of time (5 to 60 minutes) at room temperature (Fig. 1 b). The films were then separated from the tissue sections, washed in water and the residual DNA stained with toluidine blue. The tissue sections were fixed in formaldehyde, washed in water and also stained with toluidine blue. Unstained areas were observed in the parts of the films exposed to tissue sections (Fig. 1 c).
GELATINE - GLYCEROL GLASS SLIDE
GLASS SLIDE ~~sA$N~E-C~~~NFILM
(23
GELATINE -GLYCEROL GLASS SUDE
Fig. l.-Localization of DNAase in tissue sections by the substrate fiim method; (a) materials used in their relative positions before exposing the DNA film to tissue sections, (b) cross section of the same elements during exposure and (c) DNA film and tissue sections after separation and staining with toluidine blue.
@
~~~s!i!gE,~N
Fig. 2.-DNA films exposed to control and DNAase solutions and stained with toluidine blue. Experimental
Cell Research, Suppl. 7
Fig. 3.-DNA
film exposed to sections of small intestine
shown in Fig. 4. x 2.
The control studies carried out on DNA films may be summarized as follows. The fixed gelatine-DNA films remained intact in water and control solutions while a progressive loss of DNA was observed in films standing in DNAase solution (Fig. 2). Similar films exposed to blood serum became progressively toluidine-blue negative thus duplicating the results obtained with the DNAase solution. Exposure of films to tissue sections resulted in a loss of DNA in those regions in contact with tissue sections (Figs. 3 and 4) while no effect was observed in films exposed to inactivated tissues (90°C for 10 minutes). So far as the supporting medium is concerned, no effect was observed in control gelatine films placed in contact with tissue sections and stained with phloxine. It was concluded from these tests that the removal of DNA in films exposed to tissue sections was due to the hydrolytic action of tissue DNAase and the method appeared reliable for studying the distribution of this enzyme in tissue sections. In the small intestine of the rat (Figs. 5 and 6), DNAase activity was found mainly in the lumen between the villi as shown by the white (toluidine-blue negative) pattern left in the film. The epithelial cells of the villi and the crypts of Lieberktihn were also active. Presumably, DNAase is contained in the goblet cells of the villi and crypt epithelia and is discharged into the lumen together with the mucus. Weak reactions were given by the lamina propria, the submucosa and the muscle layers. In films exposed to sections of the pancreas, a reaction pattern corresponding to the acinar cells was obtained. This suggests that the pancreatic DNAase which reacts with labeled antibodies [7]. or part at least of the protein, represents the active form of the enzyme. In the thyroid (Figs. 7 and 8), DNAase activity was found mainly in the colloid, the follicular cells themselves being relatively inactive. Positive reactions were also given by the interfolicular tissue. Experimental
Cell Research, Suppl. 7
Substrate film method
Fig. B.-DNA Fig. ‘I.-DNA
43
film exposed to section of small intestine shown in Fig. 6. x 40. film exposed to section of the thyroid shown in Fig. 8. x 40.
DISTRIBUTION IN
RAT
LIVER
DURING
OF DNAaee CARCINOGENESIS
Following the examination of DNAase distribution in a few normal tissues, the DNA film method was applied to the investigation of a particular problem, namely the distribution of DNAase in rat liver during carcinogenesis by the azo-dye 4-dimethylaminoazobenzene, or DAB [5]. The DNAase activity of normal, cirrhotic and neoplastic livers had been determined previously in our laboratory by biochemical assays on tissue homogenates and slight differences only had been observed between the enzymatic activities of these tissues [5, 61. The biochemical data, however, represent mean activities of several cell types and different extracellular fluids and do not give any insight into the changes which may take place in particular tissue elements [l, 3, 41. Thus, to obtain information on the DNAase activity of the liver structural Experimental
Cell Research, Suppl. 7
R. Daousf units during carcinogenesis, the problem was investigated by histochemicar analysis. DNA films exposed to normal liver (Figs. 9 and 10) showed a positive reacSlight DNAase activity was also tion corresponding to liver parenchyma. observed in the blood vessels indicated by arrows in the same figures. In cirrhotic liver, intense DNAase activity was found in the hyperplastic parenchymal nodules (Figs. 11 and 12). IThe trabeculae composed of bile ducts and connective tissue elements were relatively inactive. The multifocal reaction observed in the trabeculae probably corresponds to groups of parenchymal cells scattered among the bile ducts and connective tissue. A peculiar feature observed in cirrhotic liver was the fact that some hyperplastic nodules were partly positive and partly negative (Figs. 13 and 14). It appeared especially interesting to note that such inactive regions of cirrhotic livers were composed of parenchymal cells which, on the basis of their architectural arrangement and basophilic properties, are often considered as the sites of origin of liver tumors. The fiber tumors (hepatomas), for their part, showed negligible DNAase activity compared with the surrounding non-tumoral tissue (Figs. 15 and 16). The weak reaction observed in the tumor.mass corresponds to trabeculae of bile duct cells and connective tissue. A further example of the low DNAase activity of the tumor cells is presented in Figs. 17 and 18 (lower part). The necrotic regions of the tumors, on the other hand, were highly active (upper parts and bottom left, corners of the same figures). The most interesting finding in this study was the fact that groups of parenchymal cells lost their DNAase activity in cirrhotic liver and that the tumor cells were practically inactive. This problem is being ‘further investigated. Attempts are also being made in this laboratory to determine whether the loss of DNAase activity by liver cells is an important factor in-tumor formation. LOCALIZATION
OF
RNAase
ACTIVITY
IN’
TISSUE
SECTIONS
During the past year, in collaboration with Mrs. H. Amano, attempts have been made to adapt the substrate film method to the localization of a second enzyme, RNAase, which hydrolyses ribonucleic acid (RNA). The procedure used at first for localizing RNAase was exactly the same as Fia. Fig. Fig. Fig. Fig.
9.-DNA film exuosed to section of normal liver shown in Fig. 10. d30. ll.-DNA film exposed to section of cirrhotic liver shown in kg. 12. x 30. 13.-DNA film exposed to section of cirrhotic liver shown in Fig. 14. x 30. 15.-DNA film exposed to section of liver tumor (hepatoma) shiwn in Fig. 16. x 30. 17.-DNA film exposed to section of necrotic liver tumor (hepatoma) shown in Fig. 18. x 30.
Experimental
Cell Research, Suppl. 7
Substrate film method
Experimental
45
Cell Research, Suppl. 7
R. Daoust that followed for DNAase except that RNA was utilized in the film instead of DNA. The fixed gelatine-RNA films exposed to control solutions were found to remain intact while a progressive loss of RNA resulted from treatment by a RNAase solution (Fig. 19). The other control studies carried out with these films gave the same results as those obtained with the DNA films. The RNA films exposed to tissue sections revealed the sites of RNAase activity in the latter and the resolution was comparable with that obtained for DNAase. In order to increase the resolution of the method, however, a different technique of film preparation was tried. Instead of preparing the films by simply spreading one drop of the gelatine-substrate mixture on the glass slide (spreading technique), 3-4 drops of the mixture were rapidly spread on the slide and the excess allowed to drain by holding the slide vertically on a piece of filter paper (draining technique). The films prepared in this way were much thinner than those obtained by the former technique and, as expected, permitted the localization of RNAase in tissue sections with a much higher resolution. The RNAase distributions reported below are examples of the results obtained with the thinner films. In the small intestine of the rat (Fig. 20) RNAase activity was especially concentrated in the lamina propria and the submucosa. Appreciable reactions were also given by the epithelial cells of the villi and the crypts of Lieberktihn. The muscle layers, on the other hand, were relatively inactive. The distribution of RNAase in the large intestine (Fig.121) was similar to that observed in the small intestine. High RNAase activities were observed in the lamina propria and the submucosa and the epithelial cells were also active. The muscularis mucosae was relatively inactive as well as the muscle layers. Fig. 21 shows an additional structure, a lymphatic nodule, which is negative. In the liver (Fig. 22), a general reaction was observed corresponding to the parenchyma while slight RNAase activity was found in the blood vessels. In the pancreas (Fig. 23), the acinar cells gave a positive reaction and some activity was also present in the intercellular spaces. The Islets of Langerhans (arrows) were relatively inactive. The Islets of Langerhans do not react either with RNAase antibodies (J. M. Marshall, personal communication) indicating that RNAase is practically absent (rather than inhibited) in these locations. POSSIBLE
APPLICATIONS
OF THE
SUBSTRATE
FILM
METHOD
The film methods for DNAase and RNAase can, in their present state, be applied to problems of several types and there is no doubt that future improvements of the method will widen the scope of the present approach. Experimental
Cell Research, Suppl. 7
Substrate jilm method
Fig. Fig. Fig. Fig.
20.-RNA 21.--RNA 22.-RNA 23.-RNA
film film film film
exposed exposed exposed exposed
to to t; to
section section section section
of of of of
47
small intestine. x 60. large intestine. x 60. liver. x 30. pancreas. x 30. Experimental
Cell Research, Suppl. 7
48
R. Daousf
Before concluding, let us esamine method seems to offer: Localization
of several different
briefly
the possibilities
the substrate
film
enzymes in tissue sections
It is probable that suitable films of a variety of substrates can be prepared and, provided that the products of the reactions can be distinguished from the substrates by staining or other procedures, such films could be applied to the localization of many different enzymes in tissues. Distribution
of enzyme activity
in fresh and fixed tissues
The films of substrate being applicable to fresh or fixed tissue sections, the method would appear especially convenient for examining the effect of fixation and other histological procedures on the activity and distribution of enzymes in tissue sections. Comparison
of enzymes in pure solutions,
biological
fluids and tissue sections
The films of substrate may be exposed to enzyme solutions, biological fluids and tissue sections, and may be useful for the comparison of the properties of tissue enzymes with those of purified enzymes. Enzyme activity
of isolated cells, nuclei and mitochondria
Films of substrate could be placed in contact with smears of isolated cells, nuclei or cell particles for studying the intracellular distribution of enzymes; the results obtained with such smears might also be compared with those obtained with sections of whole cells, nuclei or cell particles suspended in gelatine for studying the diffusion of enzymes through cell, nuclear and mitochondrial membranes. Coupling
of the substrate film method with the fluorescent antibody
method
The substrate film method localizes the active enzymes of tissues while the fluorescent antibody reaction localizes the enzyme proteins; comparing the results obtained by. these different methods might permit localization of inactive enzymes in tissue sections. Coupling
of the substrate film method with the precipitation
method
The substrate film method presumably localizes the enzymes acting at the pH of the cells while the precipitation method may reveal the distribution of enzymes at various pH values and in the presence of inhibitors or activaExperimental
Cell Research, Suppl. 7
Substrate film method
49
tom. Comparing the results obtained with such different methods might allow the localization of inhibited or activated enzymes in tissue sections. Thus, the substrate film method seems to offer many interesting possibilities for histochemical studies on enzymes. Efforts, at the moment, are still mainly concentrated on improving the method but it is also hoped to investigate soon some of its possible applications. Evidently, it is only when more results will have been obtained that one will be able to evaluate the scope of this present approach. SUMMARY Studies on the localization of enzyme activities in tissue sections by the substrate film method are described. Results are presented on the distribution of deoxyribonuclease and ribonuclease activities in sections of normal and pathological tissues and possible applications of the general method were discussed. I wish to thank Dr. A. Cantero, Director of .the Research Laboratories, Montreal Cancer Institute, for his continued interest in the present studies. Part of the work described in this paper was carried out at the Chester Beatty Research Institute in London and I am very grateful to Professor A. Haddow for his cooperation and interest. REFERENCES 1. DAOUST, R., J. Natl. Cancer Inst. 15, 1447 (1955). 2. Expfl. Cell Research 12, 203 (1957). 3. in Brauer, R., ed., Liver Function, p. 3. Am. Inst. Biol. SC., Washington, 1958: 4. Acta Unio. Intern. Contra Cancrum, in press. 5. DAOUST, R. and CANTERO, A., J. Histochkm. ‘Cytochem. 7, 139 (1959). 6. LAMIRANDE,G. DE, ALLARD, C. and CANTERO, A., Canad. J. Biochem. Physiol. 32, 35 (1954). 7. MARSHALL, J. M., Exptl. Cell Research 6 240 (1954).
4- 593710
Experimental
Cell Research, Suppl. 7