Journal of Immunological Methods, 10 (1976) 379--383
379
© North-Holland Publishing Company, Amsterdam -- Printed in The Netherlands
AUTORADIOGRAPHY AND IMMUNOHISTOPEROXIDASE TECHNIQUES APPLIED TO THE SAME TISSUE SECTION
N. VAN ROOIJEN and J.G. STREEFKERK
Department of Histology, Free University, Van der Boechorststraat 7, Amsterdam, The Netherlands (Received 7 October 1975, accepted 1 November 1975)
A method is described which allows simultaneous detection of two different compounds in tissue sections by combining autoradiography and immunohistoperoxidase techniques.
INTRODUCTION
Methods have been described for the separate detection of two labelled compounds in tissue sections or cell smears. Two different radioisotopes were used which were distinguished by double isotope autoradiography (Van Rooijen, 1973a,b, 1974, 1975). Other authors combined autoradiography and immunofluorescence for this purpose (McDevitt et al., 1966; Henderson and Smithyman, 1974). A disadvantage of the first method is the length of time required to complete the procedure: at least 3--4 months when 131I and 3 H are applied and even more than one year when 125I and 3 H are used. Moreover, high doses of radioisotopes have to be used because of the relatively low efficiency of grain production in double isotope autoradiography. A disadvantage of the second method is that fluorescent compounds and autoradiographic silver grains cannot be seen simultaneously but need two different light sources and a fluorescence microscope. Histological details in the tissues used for immunofluorescence cannot be distinguished easily. The comparison between the two labelling patterns in both methods is made either in photographs or by keeping one pattern in mind while observing the other pattern. This applies also to a recently published method for combination of immunofluorescence and immunoperoxidase techniques on the same section (Dal Canto et al., 1975). The present paper describes a method which allows simultaneous detection of two different compounds in tissue sections by combining autoradiography and immunohistoperoxidase techniques. By a relatively simple procedure, localization patterns of both compounds can be studied simultaneously in the same section with a light microscope. Autoradiography and immunohistoperoxidase techniques are nowadays commonly applied research tech-
380 niques used to detect various c o m p o u n d s or cells in tissue sections. The m e t h o d was evaluated by studying the simultaneous localization of ~2SI-HSA--anti-HSA complexes and specific antibody against horse radish peroxidase (HRP) in spleens of rats.
MATERIALS AND METHODS
Immune complexes H u m a n serum albumin (HSA; Pentex, Kankakee, Ill.) was labelled with 12sI (IMS-30 from the Radiochemical Centre, Amersham, Great Britain) using the Chloramine-T method. Immune complexes were prepared by adding rabbit anti-HSA serum ( R A H u / A l b from Nordic, Tilburg, the Netherlands) to the ~2SI-HSA.
Antigen Horse radish peroxidase (HRP; t y p e VI, Sigma, St. Louis, Mo.) was used as antigen.
Experimental design Young male Wistar rats were immunized by subcutaneous injection in the two hind f o o t pads of 150 pg HRP in 0.05 ml 0.9% NaC1 mixed with 0.05 ml complete Freund's adjuvant. They were boosted after two months by 50 pg HRP in the tail vein and 20 pg in both hind foot pads. Three days later they were injected intravenously with 10 pg I~SI-HSA (200 pCi) mixed with 0.2 ml rabbit anti-HSA-serum. The rats were killed at 2 h, 1 day or 4 days after the last injection. Blocks of spleen tissue were frozen in liquid nitrogen. Cryostat sections of 10 pm thickness were made and m o u n t e d on slides.
Detection of anti-HRP antibodies The tissue sections were treated with 100% methanol, containing 0.01% H2 O5 to minimize endogenous peroxidase activity (Streefkerk, 1972}. After rinsing three times with saline they were incubated in a HRP solution (50 pg/ ml saline) for 20 min, rinsed in saline and stained (6 min, r o o m temperature) for peroxidase activity with 3,3'-diaminobenzidine tetraHCl (Sigma; 5 mg/10 ml 0.05 M Tris--HC1 buffer) containing 0.01% H2 0 2 . A number of sections was stained with 3-amino-9~thylcarbazole (Graham et al., 1965). During the HRP-incubation and the staining procedure the slides were kept horizontal and gently shaken.
381
Autoradiography After the immunoperoxidase treatment, sections were covered with Kodak A R 10 stripping film, exposed for 6 weeks, developed, fixed, rinsed in cold water for 1 h and dried in the air. It was n o t necessary to stain the sections, because tissues showed a light background staining due to the peroxidase cytochemistry. RESULTS AND DISCUSSION
Localization of the 12SI-HSA--anti-HSA complexes The intravenously injected '25I-HSA--anti-HSA complexes were localized in the periphery of the spleen follicles at 2 h after administration; they were f o u n d in the central follicle areas at 1 and 4 days. Thus, the localization of labelled immune complexes in rats did n o t differ from that in rabbits and mice (Van Rooijen, 1973b, 1974).
Localization of anti-HRP antibody The peroxidase method used in the present study detects "specific antiHRP antibody in tissue sections (Streefkerk, 1972). Most of this antibody was present in antibody forming cells. These were found in the periphery of the periarteriolar l y m p h o c y t e sheaths and in the red pulp. In addition, anti-HRP antibody was detected in the follicle centres one or more days after the booster injection with HRP; it was visible as irregular strands and thin branched fibers. This localization pattern closely resembles that of labelled immune complexes trapped in follicle centres (Streefkerk et al., 1972). This strongly suggests that HRP--anti-HRP complexes were demonstrated which possessed free antibody binding sites for HRP, which were occupied by the H R P used in the immunohistoperoxidase procedure. It was possible that the follicular non-cellular HRP-activity visualized after the immunoperoxidase reaction, represented remnants of the intravenously injected HRP, b u t if the HRP incubation of the sections was omitted, no staining of either follicular or intraceUular H R P occurred. This indicates that both types of staining are due to anti-HRP antibody. It has been discussed earlier that follicular trapping of antigen alone or specific antibody alone does not occur (Van Rooijen, 1973b). Localization in the periphery of the follicles followed by migration towards the follicle centres takes place as soon as complexes have been formed (Van Rooijen, 1973b).
Combined autoradiography and peroxidase cytochemistry The localization of cells containing specific anti-HRP antibody was completely different from that of labelled HSA--anti-HSA complexes. This is in
382
Fig. 1. Combined autoradiographic and immunohistochemical visualization of two different compounds in rat spleen. Anti-HRP antibody present in cells (asterisks), and bound to follicular cells (arrows) is demonstrated by means of a direct immunoperoxidase procedure. Afterwards, the sections were covered with Kodak AR 10 stripping film, exposed, and developed for detection of 12SI-HSA--anti-HSA complexes. Anti-HRP antibody deposits have a brown colour, whereas the 12SI-HSA--anti-HSA complexes are represented by black grains. Therefore, microscopial distinction is easier by eye than in black and white photographs, a) Peripheral follicular localization of 12sI-HSA--anti-HSA, and more central pattern of anti-HRP, b) Magnification of a part of the follicle shown in fig. la.
a c c o r d a n c e w i t h M c D e v i t t et al. ( 1 9 6 6 ) w h o c o m b i n e d a u t o r a d i o g r a p h y a n d i m m u n o f l u o r e s c e n c e t o s t u d y t h e l o c a l i z a t i o n o f antigen in r e l a t i o n t o specific a n t i b o d y - p r o d u c i n g cells. As discussed a b o v e , p a r t o f t h e a n t i - H R P a n t i b o d y is p r e s e n t in a c o m p l e x ed f o r m w i t h H R P in t h e follicle centres. T h e l o c a l i z a t i o n p a t t e r n o f t h e s e H R P - - a n t i - H R P c o m p l e x e s was similar to t h a t o f 12SI-HSA---anti-HSA c o m p l e x e s in spleens t a k e n a f t e r an interval a f t e r t h e last i n j e c t i o n w i t h labelled i m m u n e c o m p l e x e s l o n g e n o u g h t o p e r m i t t h e c o m p l e x e s t o r e a c h t h e follicle c e n t r e s (e.g. 4 days). T w o h o u r s a f t e r i n j e c t i o n , t h e s e c o m p l e x e s w e r e still localized in t h e p e r i p h e r a l p a r t o f t h e follicles, while at this t i m e H R P - a n t i - H R P c o m p l e x e s h a d a l r e a d y r e a c h e d t h e follicle centres, f o l l o w i n g t h e ( b o o s t e r ) i n j e c t i o n o f H R P given 3 d a y s earlier t o animals w h i c h a l r e a d y h a d p r o d u c e d a n t i - H R P a n t i b o d y (fig. 1). S o m e areas in t h e follicles, w h e r e t h e c e n t r a l l o c a l i z a t i o n p a t t e r n o f t h e HRP--anti-HRP complexes overlapped the peripheral distribution of the 12SI-HSA--anti-HSA c o m p l e x e s w e r e d o u b l e labelled.
383
Evaluation o f the method In contrast with earlier published methods for separate detection by light microscopy of 2 different antigens in tissue sections (Van Rooijen, 1973; Henderson and Smithyman, 1974), the present method allows really simultaneous detection in the same section. The method combines peroxidase cytochemistry and autoradiography on light microscopic level. The black grains resulting from the autoradiographic procedures and the brownish stained sites (or reddish if amino ethylcarbazole is used for HRP staining) resulting from the peroxidase cytochemistry are readily distinguishable. In earlier methods for double isotope autoradiography or methods combining autoradiography and immunofluorescence, patterns of localization of 2 different compounds had to be observed sequentially, making exact comparison difficult. The only method of simultaneously observing the 2 patterns was to make double exposure photographs as was done by McDevitt et al. (1966); but photographs do not show all details seen under the microscope and do not allow a rapid scanning of the entire section. Combination of autoradiography and peroxidase cytochemistry has been recently used in an electron microscopic study of cell suspensions by Gonatas et al. (1974). Both auroradiography (Rogers, 1973) and immunohistoperoxidase techniques (Sternberger, 1974) have many applications in immunology. The present simple method of combining the two techniques on the same tissue section may be useful in studies distinguishing the localization of different antigens, antibodies or cells. REFERENCES Dal Canto, M.C., N.R. Blum and A.B. Johnson, 1975, J. Histochem. Cytochem. 23,452. Gonatas, N.K., A. Stieber, J. Gonatas, P. Gambetti, J.C. Antoine and S. Avrameas, 1974, J. Histochem. Cytochem. 22, 999. Graham, R.C. Jr., U. Lundholm and M.J. Karnovsky, 1965, J. Histochem. Cytochem. 13, 150. Henderson, D.C. and A.M. Smithyman, 1974, J. Immunol. Methods 6, 115. McDevitt, H.O., B.A. Askonas, J.H. Humphrey, I. Schechter and M. Sela, 1966, Immunology 11,337. Rogers, A.W., 1973, Techniques of autoradiography (Elsevier, Amsterdam). Sternberger, L.A., 1974, Immunocytochemistry (Prentice-Hall, Inc., Englewood Cliffs, N.J.). Streefkerk, J.G., 1972, J. Histochem. Cytochem. 20, 829. Streefkerk, J.G., G.A. de Graaf and R.A. de Vries, 1972, Acta Morphol. Neerl. Scand. 10, 398. Rooijen, N. van, 1973a, J. Immunol. Methods 2, 197. Rooijen, N. van, 1973b, Immunology 25,853. Rooijen, N. van, 1974, Immunology 27,617. Rooijen, N. van, 1975, J. Immunol. Methods 8,151.
379
© North-Holland Publishing Company, Amsterdam -- Printed in The Netherlands
AUTORADIOGRAPHY AND IMMUNOHISTOPEROXIDASE TECHNIQUES APPLIED TO THE SAME TISSUE SECTION
N. VAN ROOIJEN and J.G. STREEFKERK
Department of Histology, Free University, Van der Boechorststraat 7, Amsterdam, The Netherlands (Received 7 October 1975, accepted 1 November 1975)
A method is described which allows simultaneous detection of two different compounds in tissue sections by combining autoradiography and immunohistoperoxidase techniques.
INTRODUCTION
Methods have been described for the separate detection of two labelled compounds in tissue sections or cell smears. Two different radioisotopes were used which were distinguished by double isotope autoradiography (Van Rooijen, 1973a,b, 1974, 1975). Other authors combined autoradiography and immunofluorescence for this purpose (McDevitt et al., 1966; Henderson and Smithyman, 1974). A disadvantage of the first method is the length of time required to complete the procedure: at least 3--4 months when 131I and 3 H are applied and even more than one year when 125I and 3 H are used. Moreover, high doses of radioisotopes have to be used because of the relatively low efficiency of grain production in double isotope autoradiography. A disadvantage of the second method is that fluorescent compounds and autoradiographic silver grains cannot be seen simultaneously but need two different light sources and a fluorescence microscope. Histological details in the tissues used for immunofluorescence cannot be distinguished easily. The comparison between the two labelling patterns in both methods is made either in photographs or by keeping one pattern in mind while observing the other pattern. This applies also to a recently published method for combination of immunofluorescence and immunoperoxidase techniques on the same section (Dal Canto et al., 1975). The present paper describes a method which allows simultaneous detection of two different compounds in tissue sections by combining autoradiography and immunohistoperoxidase techniques. By a relatively simple procedure, localization patterns of both compounds can be studied simultaneously in the same section with a light microscope. Autoradiography and immunohistoperoxidase techniques are nowadays commonly applied research tech-
380 niques used to detect various c o m p o u n d s or cells in tissue sections. The m e t h o d was evaluated by studying the simultaneous localization of ~2SI-HSA--anti-HSA complexes and specific antibody against horse radish peroxidase (HRP) in spleens of rats.
MATERIALS AND METHODS
Immune complexes H u m a n serum albumin (HSA; Pentex, Kankakee, Ill.) was labelled with 12sI (IMS-30 from the Radiochemical Centre, Amersham, Great Britain) using the Chloramine-T method. Immune complexes were prepared by adding rabbit anti-HSA serum ( R A H u / A l b from Nordic, Tilburg, the Netherlands) to the ~2SI-HSA.
Antigen Horse radish peroxidase (HRP; t y p e VI, Sigma, St. Louis, Mo.) was used as antigen.
Experimental design Young male Wistar rats were immunized by subcutaneous injection in the two hind f o o t pads of 150 pg HRP in 0.05 ml 0.9% NaC1 mixed with 0.05 ml complete Freund's adjuvant. They were boosted after two months by 50 pg HRP in the tail vein and 20 pg in both hind foot pads. Three days later they were injected intravenously with 10 pg I~SI-HSA (200 pCi) mixed with 0.2 ml rabbit anti-HSA-serum. The rats were killed at 2 h, 1 day or 4 days after the last injection. Blocks of spleen tissue were frozen in liquid nitrogen. Cryostat sections of 10 pm thickness were made and m o u n t e d on slides.
Detection of anti-HRP antibodies The tissue sections were treated with 100% methanol, containing 0.01% H2 O5 to minimize endogenous peroxidase activity (Streefkerk, 1972}. After rinsing three times with saline they were incubated in a HRP solution (50 pg/ ml saline) for 20 min, rinsed in saline and stained (6 min, r o o m temperature) for peroxidase activity with 3,3'-diaminobenzidine tetraHCl (Sigma; 5 mg/10 ml 0.05 M Tris--HC1 buffer) containing 0.01% H2 0 2 . A number of sections was stained with 3-amino-9~thylcarbazole (Graham et al., 1965). During the HRP-incubation and the staining procedure the slides were kept horizontal and gently shaken.
381
Autoradiography After the immunoperoxidase treatment, sections were covered with Kodak A R 10 stripping film, exposed for 6 weeks, developed, fixed, rinsed in cold water for 1 h and dried in the air. It was n o t necessary to stain the sections, because tissues showed a light background staining due to the peroxidase cytochemistry. RESULTS AND DISCUSSION
Localization of the 12SI-HSA--anti-HSA complexes The intravenously injected '25I-HSA--anti-HSA complexes were localized in the periphery of the spleen follicles at 2 h after administration; they were f o u n d in the central follicle areas at 1 and 4 days. Thus, the localization of labelled immune complexes in rats did n o t differ from that in rabbits and mice (Van Rooijen, 1973b, 1974).
Localization of anti-HRP antibody The peroxidase method used in the present study detects "specific antiHRP antibody in tissue sections (Streefkerk, 1972). Most of this antibody was present in antibody forming cells. These were found in the periphery of the periarteriolar l y m p h o c y t e sheaths and in the red pulp. In addition, anti-HRP antibody was detected in the follicle centres one or more days after the booster injection with HRP; it was visible as irregular strands and thin branched fibers. This localization pattern closely resembles that of labelled immune complexes trapped in follicle centres (Streefkerk et al., 1972). This strongly suggests that HRP--anti-HRP complexes were demonstrated which possessed free antibody binding sites for HRP, which were occupied by the H R P used in the immunohistoperoxidase procedure. It was possible that the follicular non-cellular HRP-activity visualized after the immunoperoxidase reaction, represented remnants of the intravenously injected HRP, b u t if the HRP incubation of the sections was omitted, no staining of either follicular or intraceUular H R P occurred. This indicates that both types of staining are due to anti-HRP antibody. It has been discussed earlier that follicular trapping of antigen alone or specific antibody alone does not occur (Van Rooijen, 1973b). Localization in the periphery of the follicles followed by migration towards the follicle centres takes place as soon as complexes have been formed (Van Rooijen, 1973b).
Combined autoradiography and peroxidase cytochemistry The localization of cells containing specific anti-HRP antibody was completely different from that of labelled HSA--anti-HSA complexes. This is in
382
Fig. 1. Combined autoradiographic and immunohistochemical visualization of two different compounds in rat spleen. Anti-HRP antibody present in cells (asterisks), and bound to follicular cells (arrows) is demonstrated by means of a direct immunoperoxidase procedure. Afterwards, the sections were covered with Kodak AR 10 stripping film, exposed, and developed for detection of 12SI-HSA--anti-HSA complexes. Anti-HRP antibody deposits have a brown colour, whereas the 12SI-HSA--anti-HSA complexes are represented by black grains. Therefore, microscopial distinction is easier by eye than in black and white photographs, a) Peripheral follicular localization of 12sI-HSA--anti-HSA, and more central pattern of anti-HRP, b) Magnification of a part of the follicle shown in fig. la.
a c c o r d a n c e w i t h M c D e v i t t et al. ( 1 9 6 6 ) w h o c o m b i n e d a u t o r a d i o g r a p h y a n d i m m u n o f l u o r e s c e n c e t o s t u d y t h e l o c a l i z a t i o n o f antigen in r e l a t i o n t o specific a n t i b o d y - p r o d u c i n g cells. As discussed a b o v e , p a r t o f t h e a n t i - H R P a n t i b o d y is p r e s e n t in a c o m p l e x ed f o r m w i t h H R P in t h e follicle centres. T h e l o c a l i z a t i o n p a t t e r n o f t h e s e H R P - - a n t i - H R P c o m p l e x e s was similar to t h a t o f 12SI-HSA---anti-HSA c o m p l e x e s in spleens t a k e n a f t e r an interval a f t e r t h e last i n j e c t i o n w i t h labelled i m m u n e c o m p l e x e s l o n g e n o u g h t o p e r m i t t h e c o m p l e x e s t o r e a c h t h e follicle c e n t r e s (e.g. 4 days). T w o h o u r s a f t e r i n j e c t i o n , t h e s e c o m p l e x e s w e r e still localized in t h e p e r i p h e r a l p a r t o f t h e follicles, while at this t i m e H R P - a n t i - H R P c o m p l e x e s h a d a l r e a d y r e a c h e d t h e follicle centres, f o l l o w i n g t h e ( b o o s t e r ) i n j e c t i o n o f H R P given 3 d a y s earlier t o animals w h i c h a l r e a d y h a d p r o d u c e d a n t i - H R P a n t i b o d y (fig. 1). S o m e areas in t h e follicles, w h e r e t h e c e n t r a l l o c a l i z a t i o n p a t t e r n o f t h e HRP--anti-HRP complexes overlapped the peripheral distribution of the 12SI-HSA--anti-HSA c o m p l e x e s w e r e d o u b l e labelled.
383
Evaluation o f the method In contrast with earlier published methods for separate detection by light microscopy of 2 different antigens in tissue sections (Van Rooijen, 1973; Henderson and Smithyman, 1974), the present method allows really simultaneous detection in the same section. The method combines peroxidase cytochemistry and autoradiography on light microscopic level. The black grains resulting from the autoradiographic procedures and the brownish stained sites (or reddish if amino ethylcarbazole is used for HRP staining) resulting from the peroxidase cytochemistry are readily distinguishable. In earlier methods for double isotope autoradiography or methods combining autoradiography and immunofluorescence, patterns of localization of 2 different compounds had to be observed sequentially, making exact comparison difficult. The only method of simultaneously observing the 2 patterns was to make double exposure photographs as was done by McDevitt et al. (1966); but photographs do not show all details seen under the microscope and do not allow a rapid scanning of the entire section. Combination of autoradiography and peroxidase cytochemistry has been recently used in an electron microscopic study of cell suspensions by Gonatas et al. (1974). Both auroradiography (Rogers, 1973) and immunohistoperoxidase techniques (Sternberger, 1974) have many applications in immunology. The present simple method of combining the two techniques on the same tissue section may be useful in studies distinguishing the localization of different antigens, antibodies or cells. REFERENCES Dal Canto, M.C., N.R. Blum and A.B. Johnson, 1975, J. Histochem. Cytochem. 23,452. Gonatas, N.K., A. Stieber, J. Gonatas, P. Gambetti, J.C. Antoine and S. Avrameas, 1974, J. Histochem. Cytochem. 22, 999. Graham, R.C. Jr., U. Lundholm and M.J. Karnovsky, 1965, J. Histochem. Cytochem. 13, 150. Henderson, D.C. and A.M. Smithyman, 1974, J. Immunol. Methods 6, 115. McDevitt, H.O., B.A. Askonas, J.H. Humphrey, I. Schechter and M. Sela, 1966, Immunology 11,337. Rogers, A.W., 1973, Techniques of autoradiography (Elsevier, Amsterdam). Sternberger, L.A., 1974, Immunocytochemistry (Prentice-Hall, Inc., Englewood Cliffs, N.J.). Streefkerk, J.G., 1972, J. Histochem. Cytochem. 20, 829. Streefkerk, J.G., G.A. de Graaf and R.A. de Vries, 1972, Acta Morphol. Neerl. Scand. 10, 398. Rooijen, N. van, 1973a, J. Immunol. Methods 2, 197. Rooijen, N. van, 1973b, Immunology 25,853. Rooijen, N. van, 1974, Immunology 27,617. Rooijen, N. van, 1975, J. Immunol. Methods 8,151.
