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Localization of deoxyribonuclease in tissue sections
Experimental
203
Cell Research 12, 203-211 (1957)
LOCALIZATION A NEW APPROACH
OF DEOXYRIBONUCLEASE TISSUE SECTIONS TO THE
HISTOCHEMISTRY
IN
OF ENZYMES
R. DAOUST’ \
The Chester Beatty Research Institute,
Institute of Cancer Research, Royal Cancer Hospital, London, England
Received September 3, 1956
METHODS currently used to localize enzymes in tissues consist in dipping tissue sections into a solution containing a suitable substrate and precipitating the products of reaction in a visible form. These products must adhere onto the sections with a minimum of diffusion from the sites of enzyme activity [4, 81. In the present work, a different approach was investigated. The method is to place tissue sections in contact with a film of gelatine containing a substrate and, after exposure, to stain the film for the remaining substrate. The latter should be attacked in the regions of the film overlying the areas of tissue possessing enzyme activity and retained in those parts covering inactive areas. Staining of the exposed film and comparison with the corresponding tissue sections should thus reveal the sites of enzyme activity in the tissue. Films composed of deoxyribonucleic acid (DNA) dispersed in gelatine were used here in order to localize the deoxyribonuclease (DNAase) present in the tissue sections. MATERIAL
AND
METHODS
The essential steps of the gelatine-substrate film method are illustrated in Fig. 1. Fig. la shows the mat,erials used in their relative positions immediately before exposing the gelatine-DNA film to tissue sections. Fig. 1 b represents a cross section of the same elements during exposure. Fig. 1 c shows the tissue sections and the gelatine-DNA film after separation and staining. A negative image of the reactive portions of the sections is seen in the film. Preparation of gelafine-DNA films.-A 1: I mixture of 5 per cent gelatine (commercial sheet form) and 0.2 per cent DNA2 is liquefied by heating in a water-bath. One 1 This investigation was conducted during the tenure of an Exchange Fellowship under the auspices of the British Empire Cancer Campaign and of the National Cancer Institute of Canada. Permanent address: Montreal Cancer Institute, Notre-Dame Hospital, Montreal, Canada. p Highly polymerized DNA prepared from calf thymus by Mr. E. Johns of this Institute according to the method of Kay, Simmons and Dounce [S]. 13- 573702
Experimental
Cell Research 12
R. Daoust
204
drop (ca. 0.05 ml) of the mixture is placed on a glass slide and rapidly spread over a surface of about 2.5 x 4 cm with the tip of the pipette. The slide is left on a level surface at room temperature until the gelatine-DNA gel has dried. The film is then fixed overnight in neutral 10 per cent formaldehyde to render the DNA insoluble in water. After fixation, the film is washed in three successive baths of distilled water, 15 minutes each, and allowed to dry.
----CLASS SLIDE <-CELATINE-DNA FILM :=--TISSUE SECTION --‘-GELATINE-GLYCEROL ----GLASS SLIDE
----
STAINED
DNA FILM
,---UNSTAINED AREAS CORRESPONDING TO TISSUES ON LEFT
Fig. 1. Localization of DNAase in tissue sections by the gelatine-substrate film method. (a, top), materials used in their relative positions immediately before exposing the gelatine-DNA film to tissue sections. (b, center), cross section of the same elements during exposure. (c, bottom), tissue sections and gelatine-DNA film after separation and staining. A negative image of the reactive portion of the sections is seen in the film.
Preparation of tissues.-Male albino rats (ca. 200 g) are killed and the organs excised as rapidly as possible. The organs are placed in a beaker kept on ice, washed in distilled water and blotted on filter paper. A piece of each organ is cut out with a scalpel and placed immediately on a layer of ice covering a freezing stage dipped into cracked dry ice. When the tissues are frozen, the stage is adjusted on the freezing microtome kept in an insulated box at ca. -20°C and sufficient time is allowed for the tissues to adjust to this temperature. Mounting of tissue sections on gelatine-glycerol.-When sections are ready to be cut, a mixture of gelatine and glycerol (ref. 1; final concentrations used: 7 and 40 per cent respectively) is liquefied by heating in a water-bath. About 0.3 ml of this mixture are placed on a glass slide and spread over a surface of approximateIy 2.5 x 4 cm with the tip of the pipette. The slide is left on a level surface at room temperature until gelation occurs (5-10 minutes). Frozen sections are then cut at 15 ,Uaccording to the techniques of LinderstremLang, Holter and Mogensen (as described in [3]) and the sections are slid with a fine brush from the microtome knife onto the gelatine-glycerol-covered slide. The latter is introduced into the cold box only for the time necessary to transfer the sections in order to prevent freezing of the semi-solid support. The slide carrying the tissue sections is placed on a level surface at 37°C until the gelatine-glycerol liquefies and Experimental
Cell Research 12
DNAase
in fissue sections
205
the sections spread on its surface. The slide is then transferred onto a cool level surface (room temperature) and the gelatine-glycerol support is again allowed to form a firm gel (lo-20 minutes). This procedure of allowing the support to liquefy and reset prevents the sections from adhering to the gelatine-DNA film when the slides are separated after exposure. Exposure of gelatine-DNA films to tissue sections.-The slide carrying the sections is then placed against the dry gelatine-DNA film and the two are held in close contact by pressure. After standing at room temperature for a given period of time, they are separated again. The gelatine-DNA film is washed in two successive baths of distilled water, 5 minutes each, and allowed to dry. The slide supporting the tissue sections is placed in neutral 10 per cent formaldehyde, in a horizontal position, and left in the fixative overnight. It is then washed in two successive baths of distilled water, 5 minutes each, and left aside until the gelatine-glycerol support has dried. Staining of gelatine-DNA films and tissue sections with toluidine blue.-The dry gelatine-DNA film is dipped into a 0.2 per cent solution of toluidine blue for 10 minutes. The slide is then washed in distilled water to remove excess dye, the film is allowed to dry again and directly mounted with Canada balsam. Control films (2.5 per cent gelatine) do not show any appreciable staining in the same conditions. The slide carrying the tissue sections is dipped into 1: 3 acetic alcohol for 5 minutes before staining with toluidine blue. This prevents the staining of the supporting medium which contains a higher amount of gelatine than the gelatine-DNA film. The slide is transferred directly from acetic alcohol to a 0.1 per cent solution of toluidine blue. After l-2 minutes in the latter solution, the slide is washed in distilled water to remove excess dye, allowed to dry at room temperature and mounted by adding a drop of Canada balsam and covering with a coverslip. RESULTS Effect of DNAase solution on gelatine-DNA films.-Formaldehyde-fixed gelatine-DNA films treated with a solution of DNAasel at 37°C showed a progressive loss of DNA with time (Fig. 2). While a slight effect was observed after 2 hours, treatment for 8 hours resulted in an appreciable decrease in stainability, and a complete removal of the DNA was observed after 24 hours. The control solution had no effect on the films in the same conditions. Effect of tissue sections on gelatine-DNA films.-When exposed to tissue sections, the gelatine-DNA films showed a loss of DNA in those regions in contact with sections. Fig. 3 illustrates some of the negative patterns left in a film after exposure to sections of the small intestine (Fig. 4) for 1 hour and staining with toluidine blue. The negative images corresponding very well to the sections are due to the removal of DNA which is attributed to the hydrolytic action of the DNAase contained in the tissue sections. 1 The DNAase solution was prepared by adding 0.1 mg of crystalline DNAase (Worthington Chemical Lab.) per ml of McIlvaine’s buffer at pH 6.5 containing magnesium sulfate at a final concentration of 0.005 ‘21. The control solution contained the same reagents but no DNAase.
Experimental Cell Research 12
206
R. Daousf
Examination of sections and corresponding autographs at higher magnification (using a yellow filter) reveals that the DNAase activity is shown mainly by the epithelial cell layer of both the villi and the crypts of Lieberkiihn (Figs. 5 and 6). \JTeaker reactions are given by the lamina propria, the submucosa and the muscle layers but an intense reaction, assigned to a blood vessel, is also observed in the submucosa. The reaction associated with the villi epithelium is discontinuous along the epithelial cell layer and clearly extends beyond the surface into the lumen. The presence of DNAase in the lumen is particularly evident where the spaces between the villi are relatively narrow. These spaces appear tilled with the enzyme. The areas of activity associated with the crypts of Lieberkiihn do not seem, on the other hand, to cover the entire diameter of the crypts. They rather correspond to the central par1 of the glands, i.e. the apical portion of the cells and the lumen. Presumably, the DNAase is contained in the excretory region of the goblet cells of both the crypts and the villi epithelia and is discharged into the lumen together with the mucus. For studying the reaction given by the most active parts of a tissue, autographs of higher resolution can be obtained by reducing the time of exposure. Fig. 7 shows the negative image left in a film after contact with a section of the duodenum (Fig. 8) for 30 minutes, and Fig. 9 shows the results obtained by a 10 minute exposure to a section of the ileum (Fig. 10). These autographs clearly illustrate the presence of DNAase in the spaces between the villi. Mucus secretion can be observed in the same location in the corresponding sections. Moreover, focal reactions which may be assigned to individual goblet cells can be distinguished along the region of the villi epithelium, on each side of the active areas associated with the pits (Fig. 7, left part). These individual reactions are still more evident in some autographs of the crypts of Lieberkuhn (Fig. 9, bottom left corner). to the acinar cells With the pancreas, a negative pattern corresponding was obtained (Figs. 11 and 12). A multifocal reaction was sometimes observed in adjacent acini, suggesting that the DNAase is not evenly distributed in the acini but more concentrated in their centers, i.e. in the secretory pole of the cells. In the thyroid, DNAase activity was shown by the colloid present within the follicles, the intensity of the reaction varying from one follicle to another (Figs. 13 and 14). The follicular epithelium itself was relatively inactive. The interfollicular connective tissue showed a multifocal reaction which may be due to the small blood vessels present in this location.
Experimental
Cell Research 12
DNAme in tissue sectiom
1;ig. 2. Celatine-DNA films treated with control or DNAase solutions for different periods of time and stained with toluidine blue. Fig. 3. Gelatine-DNA film exposed to sections of the small intestine for 1 hour and stained with toiuidine blue. Magnification Y 2.5. Fig. 4. Corresponding tissue sections ( ‘~ 2..5) stained
se-..------
24 hrt
c--e-w---
Fig. 5. Part of a negative image left in a gelatine-DNA film exposed to sections intestine for one hour (Fig. 3) seen at higher magnification ( x 10). Fig. 6. Corresponding tissue section at same magnification. 13t - 573702
Experimental
of the small
Cell Research 12
208
Experimental
R. Daoust
Cell Research 12
DNAase in fissue sections DISCUSSION
This first attempt to localize enzymes in tissues by the use of gelatinesubstrate films has proved successful and the new method seems to present many advantages. Firstly, fresh tissue sections can easily be used in this method, thus permitting to localize several enzymes which are inactivated or dissolved by the treatments necessary in the usual histochemical techniques [4]. Secondly, the demonstration of enzyme activity is here directly related to changes in the substrate instead of being associated with the formation of degradation products. This eliminates several problems encountered in the usual procedures such as suitable reactions for precipitating the end-products in a visible form, diffusion of these products from their sites of formation, adsorption on inactive parts of the tissue, etc. [2, 4, 5, 81. In addition, the method offers the advantage of being applicable to biological fluids. Since films of substrate can be used with solutions as well as with tissue sections, they may also find applications in related fields: studies on enzymes specificity, comparisons between cellular and purified enzymes, etc. A disadvantage of the method is its lower resolution, as compared with the end-product precipitation methods which can reveal the intracellular distribution of enzymes. A localization at that level could sometimes be achieved by the present method in special cases where the cells present a particular arrangement. The studies on the small intestine, for instance, have suggested that active DNAase is contained in the secretory region of the goblet cells and absent from their base. However, the method, in its present state at least, is not generally applicable to the cellular level. The resolution of the method depends upon several factors: the thickness of the film, the concentration of the substrate in that film, the distance between the sections and the film, and the duration of the exposure. The composition of the semi-solid
Fig. 7. Negative image left in a gelatine-DNA film exposed to sections of the duodenum for 34 minutes and stained with toluidine blue. Magnification x40. Fig. 8. Corresponding tissue section ( x 40) stained with toluidine blue. Fig. 9. Negative image left in a gelatine-DNA film exposed to sections of the ileum for 10 minutes and stained with toluidine blue. Magnification x 40. Fig. 10. Corresponding tissue section ( x 40) stained with toluidine blue. Fig. 11. Negative image left in a gelatine-DNA film exposed to sections of the pancreas for I hour and stained with toluidine blue. Magnification x 40. Fig. 12. Corresponding tissue section ( x 40) stained with toluidine blua. Fig. 13. Negative image left in a gelatine-DNA film exposed to sections of the thyroid for 5 minutes and stained with toluidine blue. Magnification x 40. Fig. 14. Corresponding tissue section ( x 40) stained with toluidine blue. Experimental
Cell Research 12
R. Daousf support for the sections is also important. It was observed that the addition of glycerol to the support greatly improved the precision of the autographic image, presumably by reducing the rate of enzyme diffusion. The results described above were obtained under the optimum conditions determined after series of assays. While the property of enzymes to diffuse through a thin film is actually the basis of the method, the optimum conditions will naturally be those which will minimize the geometrical affects of enzyme diffusion, a situation in many ways similar to that encountered in radioautography. The present approach, which has proved practical for localizing DNAase activity, is probably applicable to many different enzymes since suitable films of several substrates can likely be prepared. To visualize the unattacked substrate remaining in the film after exposure, any staining or other method can be used. This procedure does not need to be specific for the substrate, as long as the second component of the film, gelatine, does not stain by the same technique. Thus, the method seems to offer interesting advantages and should prove fruitful in various studies. In comparing methods for localizing enzyme activities in tissues, no mention was made of the use of fluorescein-labelled antibodies [7]. In that method, the reaction localizes the enzymes as proteins, i.e. does not distinguish between the inactive and the active forms of enzymes, while the methods discussed above localize selectively the active fraction of these proteins. An interesting application of the antigen-antibody reaction might be its use in conjunction with methods based on the activity of enzymes to localize, by difference, the inactive or latent form of enzymes. SUMMARY
A new approach to the localization of enzymes in tissues was investigated. The method is to place tissue sections in contact with a film of gelatine containing a substrate, to allow the tissue enzyme to act upon its substrate in the film and, after separating the sections and the film, to stain the latter for the remaining unattacked substrate. The latter is attacked in the regions of the film overlying the areas of tissue possessing enzyme activity and retained in those parts covering inactive areas. Comparing the negative pattern left in the film with the corresponding tissue sections reveals the sites of activity in the tissue. Films made of deoxyribonucleic acid dispersed in gelatine were used in order to localize deoxyribonuclease in tissue sections. The method proved Experimentat
Cell Research 12
DNAase in tissue sections satisfactory for this enzyme which was thus localized in the small intestine, pancreas and thyroid of the rat. The method appears applicable to the localization of other enzymes. I wish to thank Prof. A. Haddow for his interest in the present study, Prof. J. A. V. Butler, Dr. P. F. Davison and Mr. E. Johns for supplying the preparations of deoxyribonucleic acid, and Mr. K. G. Moreman and his staff for preparing the illustrations.
This investigation
has been supported by grants to the Chester Beatty Research
Institute (Institute of Cancer Research: Royal Cancer Hospital) from the British Empire Cancer Campaign, the Jane Coffin Childs Memorial Fund for Medical Research, the Anna Fuller Fund and the National Cancer Institute of the National Institutes of Health, U.S. Public Health Service. REFERENCES 1. BAKER, J. R., Cytological Technique. Methuen and Co., London, 1945. 2. DANIELLI, J. F., Cytochemistry. John Wiley and Sons, Inc., New York, 1953. 3. GLICK, D., Techniques of Histo- and Cytochemistry. Inter-science Publ., Inc., New York, 1949. 4. GOMORI, G., Microscopic Histochemistry. The University of Chicago Press, Chicago, 1952. 5. HOLT, S. J., Proc. Royal Sot., Ser. B, 142, 160 (1954). 6. KAY, E. R. M., SIMMONS, N. S., and DOUNCE, A. L., J. Am. Chem. Sot. 74, 1724 (1952). 7. MARSHALL, J. M., Expil. Cell Research 6, 240 (1954). 8. PEARSE, A. G. E., Histochemistry. Churchill Ltd., London, 1953.
Experimental
Cell Research 12
203
Cell Research 12, 203-211 (1957)
LOCALIZATION A NEW APPROACH
OF DEOXYRIBONUCLEASE TISSUE SECTIONS TO THE
HISTOCHEMISTRY
IN
OF ENZYMES
R. DAOUST’ \
The Chester Beatty Research Institute,
Institute of Cancer Research, Royal Cancer Hospital, London, England
Received September 3, 1956
METHODS currently used to localize enzymes in tissues consist in dipping tissue sections into a solution containing a suitable substrate and precipitating the products of reaction in a visible form. These products must adhere onto the sections with a minimum of diffusion from the sites of enzyme activity [4, 81. In the present work, a different approach was investigated. The method is to place tissue sections in contact with a film of gelatine containing a substrate and, after exposure, to stain the film for the remaining substrate. The latter should be attacked in the regions of the film overlying the areas of tissue possessing enzyme activity and retained in those parts covering inactive areas. Staining of the exposed film and comparison with the corresponding tissue sections should thus reveal the sites of enzyme activity in the tissue. Films composed of deoxyribonucleic acid (DNA) dispersed in gelatine were used here in order to localize the deoxyribonuclease (DNAase) present in the tissue sections. MATERIAL
AND
METHODS
The essential steps of the gelatine-substrate film method are illustrated in Fig. 1. Fig. la shows the mat,erials used in their relative positions immediately before exposing the gelatine-DNA film to tissue sections. Fig. 1 b represents a cross section of the same elements during exposure. Fig. 1 c shows the tissue sections and the gelatine-DNA film after separation and staining. A negative image of the reactive portions of the sections is seen in the film. Preparation of gelafine-DNA films.-A 1: I mixture of 5 per cent gelatine (commercial sheet form) and 0.2 per cent DNA2 is liquefied by heating in a water-bath. One 1 This investigation was conducted during the tenure of an Exchange Fellowship under the auspices of the British Empire Cancer Campaign and of the National Cancer Institute of Canada. Permanent address: Montreal Cancer Institute, Notre-Dame Hospital, Montreal, Canada. p Highly polymerized DNA prepared from calf thymus by Mr. E. Johns of this Institute according to the method of Kay, Simmons and Dounce [S]. 13- 573702
Experimental
Cell Research 12
R. Daoust
204
drop (ca. 0.05 ml) of the mixture is placed on a glass slide and rapidly spread over a surface of about 2.5 x 4 cm with the tip of the pipette. The slide is left on a level surface at room temperature until the gelatine-DNA gel has dried. The film is then fixed overnight in neutral 10 per cent formaldehyde to render the DNA insoluble in water. After fixation, the film is washed in three successive baths of distilled water, 15 minutes each, and allowed to dry.
----CLASS SLIDE <-CELATINE-DNA FILM :=--TISSUE SECTION --‘-GELATINE-GLYCEROL ----GLASS SLIDE
----
STAINED
DNA FILM
,---UNSTAINED AREAS CORRESPONDING TO TISSUES ON LEFT
Fig. 1. Localization of DNAase in tissue sections by the gelatine-substrate film method. (a, top), materials used in their relative positions immediately before exposing the gelatine-DNA film to tissue sections. (b, center), cross section of the same elements during exposure. (c, bottom), tissue sections and gelatine-DNA film after separation and staining. A negative image of the reactive portion of the sections is seen in the film.
Preparation of tissues.-Male albino rats (ca. 200 g) are killed and the organs excised as rapidly as possible. The organs are placed in a beaker kept on ice, washed in distilled water and blotted on filter paper. A piece of each organ is cut out with a scalpel and placed immediately on a layer of ice covering a freezing stage dipped into cracked dry ice. When the tissues are frozen, the stage is adjusted on the freezing microtome kept in an insulated box at ca. -20°C and sufficient time is allowed for the tissues to adjust to this temperature. Mounting of tissue sections on gelatine-glycerol.-When sections are ready to be cut, a mixture of gelatine and glycerol (ref. 1; final concentrations used: 7 and 40 per cent respectively) is liquefied by heating in a water-bath. About 0.3 ml of this mixture are placed on a glass slide and spread over a surface of approximateIy 2.5 x 4 cm with the tip of the pipette. The slide is left on a level surface at room temperature until gelation occurs (5-10 minutes). Frozen sections are then cut at 15 ,Uaccording to the techniques of LinderstremLang, Holter and Mogensen (as described in [3]) and the sections are slid with a fine brush from the microtome knife onto the gelatine-glycerol-covered slide. The latter is introduced into the cold box only for the time necessary to transfer the sections in order to prevent freezing of the semi-solid support. The slide carrying the tissue sections is placed on a level surface at 37°C until the gelatine-glycerol liquefies and Experimental
Cell Research 12
DNAase
in fissue sections
205
the sections spread on its surface. The slide is then transferred onto a cool level surface (room temperature) and the gelatine-glycerol support is again allowed to form a firm gel (lo-20 minutes). This procedure of allowing the support to liquefy and reset prevents the sections from adhering to the gelatine-DNA film when the slides are separated after exposure. Exposure of gelatine-DNA films to tissue sections.-The slide carrying the sections is then placed against the dry gelatine-DNA film and the two are held in close contact by pressure. After standing at room temperature for a given period of time, they are separated again. The gelatine-DNA film is washed in two successive baths of distilled water, 5 minutes each, and allowed to dry. The slide supporting the tissue sections is placed in neutral 10 per cent formaldehyde, in a horizontal position, and left in the fixative overnight. It is then washed in two successive baths of distilled water, 5 minutes each, and left aside until the gelatine-glycerol support has dried. Staining of gelatine-DNA films and tissue sections with toluidine blue.-The dry gelatine-DNA film is dipped into a 0.2 per cent solution of toluidine blue for 10 minutes. The slide is then washed in distilled water to remove excess dye, the film is allowed to dry again and directly mounted with Canada balsam. Control films (2.5 per cent gelatine) do not show any appreciable staining in the same conditions. The slide carrying the tissue sections is dipped into 1: 3 acetic alcohol for 5 minutes before staining with toluidine blue. This prevents the staining of the supporting medium which contains a higher amount of gelatine than the gelatine-DNA film. The slide is transferred directly from acetic alcohol to a 0.1 per cent solution of toluidine blue. After l-2 minutes in the latter solution, the slide is washed in distilled water to remove excess dye, allowed to dry at room temperature and mounted by adding a drop of Canada balsam and covering with a coverslip. RESULTS Effect of DNAase solution on gelatine-DNA films.-Formaldehyde-fixed gelatine-DNA films treated with a solution of DNAasel at 37°C showed a progressive loss of DNA with time (Fig. 2). While a slight effect was observed after 2 hours, treatment for 8 hours resulted in an appreciable decrease in stainability, and a complete removal of the DNA was observed after 24 hours. The control solution had no effect on the films in the same conditions. Effect of tissue sections on gelatine-DNA films.-When exposed to tissue sections, the gelatine-DNA films showed a loss of DNA in those regions in contact with sections. Fig. 3 illustrates some of the negative patterns left in a film after exposure to sections of the small intestine (Fig. 4) for 1 hour and staining with toluidine blue. The negative images corresponding very well to the sections are due to the removal of DNA which is attributed to the hydrolytic action of the DNAase contained in the tissue sections. 1 The DNAase solution was prepared by adding 0.1 mg of crystalline DNAase (Worthington Chemical Lab.) per ml of McIlvaine’s buffer at pH 6.5 containing magnesium sulfate at a final concentration of 0.005 ‘21. The control solution contained the same reagents but no DNAase.
Experimental Cell Research 12
206
R. Daousf
Examination of sections and corresponding autographs at higher magnification (using a yellow filter) reveals that the DNAase activity is shown mainly by the epithelial cell layer of both the villi and the crypts of Lieberkiihn (Figs. 5 and 6). \JTeaker reactions are given by the lamina propria, the submucosa and the muscle layers but an intense reaction, assigned to a blood vessel, is also observed in the submucosa. The reaction associated with the villi epithelium is discontinuous along the epithelial cell layer and clearly extends beyond the surface into the lumen. The presence of DNAase in the lumen is particularly evident where the spaces between the villi are relatively narrow. These spaces appear tilled with the enzyme. The areas of activity associated with the crypts of Lieberkiihn do not seem, on the other hand, to cover the entire diameter of the crypts. They rather correspond to the central par1 of the glands, i.e. the apical portion of the cells and the lumen. Presumably, the DNAase is contained in the excretory region of the goblet cells of both the crypts and the villi epithelia and is discharged into the lumen together with the mucus. For studying the reaction given by the most active parts of a tissue, autographs of higher resolution can be obtained by reducing the time of exposure. Fig. 7 shows the negative image left in a film after contact with a section of the duodenum (Fig. 8) for 30 minutes, and Fig. 9 shows the results obtained by a 10 minute exposure to a section of the ileum (Fig. 10). These autographs clearly illustrate the presence of DNAase in the spaces between the villi. Mucus secretion can be observed in the same location in the corresponding sections. Moreover, focal reactions which may be assigned to individual goblet cells can be distinguished along the region of the villi epithelium, on each side of the active areas associated with the pits (Fig. 7, left part). These individual reactions are still more evident in some autographs of the crypts of Lieberkuhn (Fig. 9, bottom left corner). to the acinar cells With the pancreas, a negative pattern corresponding was obtained (Figs. 11 and 12). A multifocal reaction was sometimes observed in adjacent acini, suggesting that the DNAase is not evenly distributed in the acini but more concentrated in their centers, i.e. in the secretory pole of the cells. In the thyroid, DNAase activity was shown by the colloid present within the follicles, the intensity of the reaction varying from one follicle to another (Figs. 13 and 14). The follicular epithelium itself was relatively inactive. The interfollicular connective tissue showed a multifocal reaction which may be due to the small blood vessels present in this location.
Experimental
Cell Research 12
DNAme in tissue sectiom
1;ig. 2. Celatine-DNA films treated with control or DNAase solutions for different periods of time and stained with toluidine blue. Fig. 3. Gelatine-DNA film exposed to sections of the small intestine for 1 hour and stained with toiuidine blue. Magnification Y 2.5. Fig. 4. Corresponding tissue sections ( ‘~ 2..5) stained
se-..------
24 hrt
c--e-w---
Fig. 5. Part of a negative image left in a gelatine-DNA film exposed to sections intestine for one hour (Fig. 3) seen at higher magnification ( x 10). Fig. 6. Corresponding tissue section at same magnification. 13t - 573702
Experimental
of the small
Cell Research 12
208
Experimental
R. Daoust
Cell Research 12
DNAase in fissue sections DISCUSSION
This first attempt to localize enzymes in tissues by the use of gelatinesubstrate films has proved successful and the new method seems to present many advantages. Firstly, fresh tissue sections can easily be used in this method, thus permitting to localize several enzymes which are inactivated or dissolved by the treatments necessary in the usual histochemical techniques [4]. Secondly, the demonstration of enzyme activity is here directly related to changes in the substrate instead of being associated with the formation of degradation products. This eliminates several problems encountered in the usual procedures such as suitable reactions for precipitating the end-products in a visible form, diffusion of these products from their sites of formation, adsorption on inactive parts of the tissue, etc. [2, 4, 5, 81. In addition, the method offers the advantage of being applicable to biological fluids. Since films of substrate can be used with solutions as well as with tissue sections, they may also find applications in related fields: studies on enzymes specificity, comparisons between cellular and purified enzymes, etc. A disadvantage of the method is its lower resolution, as compared with the end-product precipitation methods which can reveal the intracellular distribution of enzymes. A localization at that level could sometimes be achieved by the present method in special cases where the cells present a particular arrangement. The studies on the small intestine, for instance, have suggested that active DNAase is contained in the secretory region of the goblet cells and absent from their base. However, the method, in its present state at least, is not generally applicable to the cellular level. The resolution of the method depends upon several factors: the thickness of the film, the concentration of the substrate in that film, the distance between the sections and the film, and the duration of the exposure. The composition of the semi-solid
Fig. 7. Negative image left in a gelatine-DNA film exposed to sections of the duodenum for 34 minutes and stained with toluidine blue. Magnification x40. Fig. 8. Corresponding tissue section ( x 40) stained with toluidine blue. Fig. 9. Negative image left in a gelatine-DNA film exposed to sections of the ileum for 10 minutes and stained with toluidine blue. Magnification x 40. Fig. 10. Corresponding tissue section ( x 40) stained with toluidine blue. Fig. 11. Negative image left in a gelatine-DNA film exposed to sections of the pancreas for I hour and stained with toluidine blue. Magnification x 40. Fig. 12. Corresponding tissue section ( x 40) stained with toluidine blua. Fig. 13. Negative image left in a gelatine-DNA film exposed to sections of the thyroid for 5 minutes and stained with toluidine blue. Magnification x 40. Fig. 14. Corresponding tissue section ( x 40) stained with toluidine blue. Experimental
Cell Research 12
R. Daousf support for the sections is also important. It was observed that the addition of glycerol to the support greatly improved the precision of the autographic image, presumably by reducing the rate of enzyme diffusion. The results described above were obtained under the optimum conditions determined after series of assays. While the property of enzymes to diffuse through a thin film is actually the basis of the method, the optimum conditions will naturally be those which will minimize the geometrical affects of enzyme diffusion, a situation in many ways similar to that encountered in radioautography. The present approach, which has proved practical for localizing DNAase activity, is probably applicable to many different enzymes since suitable films of several substrates can likely be prepared. To visualize the unattacked substrate remaining in the film after exposure, any staining or other method can be used. This procedure does not need to be specific for the substrate, as long as the second component of the film, gelatine, does not stain by the same technique. Thus, the method seems to offer interesting advantages and should prove fruitful in various studies. In comparing methods for localizing enzyme activities in tissues, no mention was made of the use of fluorescein-labelled antibodies [7]. In that method, the reaction localizes the enzymes as proteins, i.e. does not distinguish between the inactive and the active forms of enzymes, while the methods discussed above localize selectively the active fraction of these proteins. An interesting application of the antigen-antibody reaction might be its use in conjunction with methods based on the activity of enzymes to localize, by difference, the inactive or latent form of enzymes. SUMMARY
A new approach to the localization of enzymes in tissues was investigated. The method is to place tissue sections in contact with a film of gelatine containing a substrate, to allow the tissue enzyme to act upon its substrate in the film and, after separating the sections and the film, to stain the latter for the remaining unattacked substrate. The latter is attacked in the regions of the film overlying the areas of tissue possessing enzyme activity and retained in those parts covering inactive areas. Comparing the negative pattern left in the film with the corresponding tissue sections reveals the sites of activity in the tissue. Films made of deoxyribonucleic acid dispersed in gelatine were used in order to localize deoxyribonuclease in tissue sections. The method proved Experimentat
Cell Research 12
DNAase in tissue sections satisfactory for this enzyme which was thus localized in the small intestine, pancreas and thyroid of the rat. The method appears applicable to the localization of other enzymes. I wish to thank Prof. A. Haddow for his interest in the present study, Prof. J. A. V. Butler, Dr. P. F. Davison and Mr. E. Johns for supplying the preparations of deoxyribonucleic acid, and Mr. K. G. Moreman and his staff for preparing the illustrations.
This investigation
has been supported by grants to the Chester Beatty Research
Institute (Institute of Cancer Research: Royal Cancer Hospital) from the British Empire Cancer Campaign, the Jane Coffin Childs Memorial Fund for Medical Research, the Anna Fuller Fund and the National Cancer Institute of the National Institutes of Health, U.S. Public Health Service. REFERENCES 1. BAKER, J. R., Cytological Technique. Methuen and Co., London, 1945. 2. DANIELLI, J. F., Cytochemistry. John Wiley and Sons, Inc., New York, 1953. 3. GLICK, D., Techniques of Histo- and Cytochemistry. Inter-science Publ., Inc., New York, 1949. 4. GOMORI, G., Microscopic Histochemistry. The University of Chicago Press, Chicago, 1952. 5. HOLT, S. J., Proc. Royal Sot., Ser. B, 142, 160 (1954). 6. KAY, E. R. M., SIMMONS, N. S., and DOUNCE, A. L., J. Am. Chem. Sot. 74, 1724 (1952). 7. MARSHALL, J. M., Expil. Cell Research 6, 240 (1954). 8. PEARSE, A. G. E., Histochemistry. Churchill Ltd., London, 1953.
Experimental
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