Histopathology 1977,
I,
371-374
Immunofluorescenceon resin-embedded material
J.S@OTTO Unit6 de Recherche d’Hkpatologie Injantile, INSERM U.56, Hspital d’Enfants,94270 Le Kremlin-BicEtre, France
Accepted for publication 27 June 1977 SCOTTO J. (1977) Histopathology I, 371-374
Immunofluorescence on resin-embedded material
Some antigenic determinants can be preserved in tissues after plastic embedding. In the present study liver tissue was fixed with glutaraldehyde or paraformaldehyde, dehydrated with ethanol and toluene and embedded in araldite which was then polymerized at 37°C. Immunofluorescence was performed on semi-thin sections etched with hydrogen peroxide. This procedure allows correlation with light microscopy (on the same stained semi-thin section) and with electron microscopy (on adjacent ultra-thin section). Good results were obtained with anti-nuclear, anti-mitochondrial, anti-microsomal and anti-al-antitrypsin sera. Keywords : resin-embedding, immunofluorescence, electron microscopy
Introduction
The direct localization of antigens at the ultrastructural level can be performed by two methods. The first uses labelled immunoglobulin fractions or labelled purified antibodies applied to tissues prior to embedding. In the second highly purified, labelled antibodies are applied to sectioned material (Sternberger 1967). The best morphological results have been obtained with the second method, especially in the detection of some of the pituitary hormones (Moriarty 1973). The principal difficulties vary according to the type of method. These difficulties include achievement of good permeability for the marker without undue cell damage, obtaining highly purified antibodies, preserving the antigen determinants after embedding and etching of resin to allow access of the reagents to the tissue. These technical problems limit the successful application of the methods to a few specialized laboratories. Methods for light microscopic immunofluorescence in semi-thin sections of resinembedded material have recently been developed. Comparison with adjacent ultrathin sections provides an easier way of localizing antigens at the ultrastructural level.
372
J.Scotto
This approach has been applied to the visualization of pituitary hormones (Bugnon, Lenys & Herlant 1974, Tramu, Beauvillain & Dubois 1974). The present study describes a similar method for the detection of some antigenic determinants in liver cells. Material and methods PREPARATION OF THE MATERIAL
Human and rat liver specimens were cut into I mm cubes and fixed at 4°C in a solution of 2.5 % glutaraldehyde or paraformaldehyde in 0. I M phosphate buffer at pH 7.2-7.4. This solution was continuously stirred for 4 h. Washing was performed in a constantly agitated solution of the same buffer for 3 h at 4"C, followed by renewal of buffer and over-night storage at 4°C without stirring. Specimens were dehydrated at 20'C in a graded series of ethanol solutions (70, 90% and 3 changes of absolute, each change for 20 min except the last for I h), followed by two consecutive changes of stirred toluene, each for 10min. Subsequently they were impregnated with a moving mixture of araldite and toluene in the proportions 1/3-2/3 then 2/3-1/3, each for 30 rnin and immersed in araldite without stirring for I h at 37°C. After embedding in araldite, the polymerization was carried out at 37°C until completion (3 or 4 days).
STAINING PROCEDURE
Semi-thin sections, 0.5-1 pm in thickness, were etched with 10% aqueous hydrogen peroxide for 30 min, washed in three changes of distilled water and allowed to dry. For immunofluorescence they were then covered with a drop of anti-serum at various concentrations in phosphate-buffered saline for 15 min to I h and washed in three changes of 0. I M phosphate buffer at pH 7.2-7.4 for 5 min each. In the direct method, ie. with labelled antiserum, the specimens were then mounted in buffered glycerol. In the indirect method, they were incubated for 5-15 min under a drop of labelled antigammaglobulin at suitable concentration and washed in three changes of phosphate buffer, for 15 min each, before mounting. The slides were stored at 4°C and examined within 24 h. The same sections, washed in distilled water and dried, can subsequently be stained for histological purposes with various dyes, after removal of the resin if necessary (Lane & Europa 1965). Adjacent ultrathin sections, contrasted as usual for electron microscopy, can be used for ultrastructural correlation. Results
With this method, using anti-nuclear, anti-mitochondria1 and anti-microsomal
Figure I. Structures demonstrated with relevant antisera in resin embedded sections: mitochondria (A) nuclei (B) in rat hepatocytes: nuclei (C) and microsomes or endoplasmic reticulum (D) in human hepatocytes. All x 1600.
Figure 2. Alpha-I-antitrypsin bodies demonstrated in resin-embedded sections by immunofluorescence (A), same section after PAS staining (B) and electron microscopy (C and D). XSOO (A &L B), x 1350 (C), X4500 (D). D*
374
JScotto
sera, we have been able to identify corresponding structures in rat and human liver (Figure I). Using anti-a ,-antitrypsin antibodies we were able to demonstrate the presence of an antigenically related substance in some hepatocytes of patients with CI ,-antitrypsin deficiency. This substance was then stained by the PAS method (without diastase digestion) and corresponded to amorphous material observed with electron microscopy on adjacent ultrathin sections (Figure 2).
References BUGNON C . , LENYS D. & HERLANT M. (1974) CaractCrisation de diverses cellules de I’adCnohypophyse du Renard par immunofluorescence sur coupes semi-fines et superposition des donnees de microscopie electronique. Comptes Rendus de l’dcademie des Sciences (Paris) 278, I 243-1 247 LANER.P. & EUROPAD.L. (1965) Differential staining of ultrathin sections of epon-embedded tissues for light microscopy. Journal ojHistochemistry and Cytochemistry 13, 579-582 MORIARTY G.C. (1973) Adenohypophysis : Ultrastructural cytochemistry : a review. Journal of Histochemistry und Cytochemistry 21, 855-894 STERNBERGER L.A. (1967) Electron microscopic immunocytochemistry : a review. Journal of Histochemistry and Cytochemistry 15,I 39-1 59 TRAMU G., BEAUVILLAIN J.CL. & DUBOIS M.P. (1974) Les cellules Gonadotropes de l’hypophyse du LBrot (Eliomys quercinus) : concordance des rksultats de microscopie optique (irnmunofluorescence et colorations) et de microscopie Clectronique. Joumai de Microscupie 21, I 8 1-184
I,
371-374
Immunofluorescenceon resin-embedded material
J.S@OTTO Unit6 de Recherche d’Hkpatologie Injantile, INSERM U.56, Hspital d’Enfants,94270 Le Kremlin-BicEtre, France
Accepted for publication 27 June 1977 SCOTTO J. (1977) Histopathology I, 371-374
Immunofluorescence on resin-embedded material
Some antigenic determinants can be preserved in tissues after plastic embedding. In the present study liver tissue was fixed with glutaraldehyde or paraformaldehyde, dehydrated with ethanol and toluene and embedded in araldite which was then polymerized at 37°C. Immunofluorescence was performed on semi-thin sections etched with hydrogen peroxide. This procedure allows correlation with light microscopy (on the same stained semi-thin section) and with electron microscopy (on adjacent ultra-thin section). Good results were obtained with anti-nuclear, anti-mitochondrial, anti-microsomal and anti-al-antitrypsin sera. Keywords : resin-embedding, immunofluorescence, electron microscopy
Introduction
The direct localization of antigens at the ultrastructural level can be performed by two methods. The first uses labelled immunoglobulin fractions or labelled purified antibodies applied to tissues prior to embedding. In the second highly purified, labelled antibodies are applied to sectioned material (Sternberger 1967). The best morphological results have been obtained with the second method, especially in the detection of some of the pituitary hormones (Moriarty 1973). The principal difficulties vary according to the type of method. These difficulties include achievement of good permeability for the marker without undue cell damage, obtaining highly purified antibodies, preserving the antigen determinants after embedding and etching of resin to allow access of the reagents to the tissue. These technical problems limit the successful application of the methods to a few specialized laboratories. Methods for light microscopic immunofluorescence in semi-thin sections of resinembedded material have recently been developed. Comparison with adjacent ultrathin sections provides an easier way of localizing antigens at the ultrastructural level.
372
J.Scotto
This approach has been applied to the visualization of pituitary hormones (Bugnon, Lenys & Herlant 1974, Tramu, Beauvillain & Dubois 1974). The present study describes a similar method for the detection of some antigenic determinants in liver cells. Material and methods PREPARATION OF THE MATERIAL
Human and rat liver specimens were cut into I mm cubes and fixed at 4°C in a solution of 2.5 % glutaraldehyde or paraformaldehyde in 0. I M phosphate buffer at pH 7.2-7.4. This solution was continuously stirred for 4 h. Washing was performed in a constantly agitated solution of the same buffer for 3 h at 4"C, followed by renewal of buffer and over-night storage at 4°C without stirring. Specimens were dehydrated at 20'C in a graded series of ethanol solutions (70, 90% and 3 changes of absolute, each change for 20 min except the last for I h), followed by two consecutive changes of stirred toluene, each for 10min. Subsequently they were impregnated with a moving mixture of araldite and toluene in the proportions 1/3-2/3 then 2/3-1/3, each for 30 rnin and immersed in araldite without stirring for I h at 37°C. After embedding in araldite, the polymerization was carried out at 37°C until completion (3 or 4 days).
STAINING PROCEDURE
Semi-thin sections, 0.5-1 pm in thickness, were etched with 10% aqueous hydrogen peroxide for 30 min, washed in three changes of distilled water and allowed to dry. For immunofluorescence they were then covered with a drop of anti-serum at various concentrations in phosphate-buffered saline for 15 min to I h and washed in three changes of 0. I M phosphate buffer at pH 7.2-7.4 for 5 min each. In the direct method, ie. with labelled antiserum, the specimens were then mounted in buffered glycerol. In the indirect method, they were incubated for 5-15 min under a drop of labelled antigammaglobulin at suitable concentration and washed in three changes of phosphate buffer, for 15 min each, before mounting. The slides were stored at 4°C and examined within 24 h. The same sections, washed in distilled water and dried, can subsequently be stained for histological purposes with various dyes, after removal of the resin if necessary (Lane & Europa 1965). Adjacent ultrathin sections, contrasted as usual for electron microscopy, can be used for ultrastructural correlation. Results
With this method, using anti-nuclear, anti-mitochondria1 and anti-microsomal
Figure I. Structures demonstrated with relevant antisera in resin embedded sections: mitochondria (A) nuclei (B) in rat hepatocytes: nuclei (C) and microsomes or endoplasmic reticulum (D) in human hepatocytes. All x 1600.
Figure 2. Alpha-I-antitrypsin bodies demonstrated in resin-embedded sections by immunofluorescence (A), same section after PAS staining (B) and electron microscopy (C and D). XSOO (A &L B), x 1350 (C), X4500 (D). D*
374
JScotto
sera, we have been able to identify corresponding structures in rat and human liver (Figure I). Using anti-a ,-antitrypsin antibodies we were able to demonstrate the presence of an antigenically related substance in some hepatocytes of patients with CI ,-antitrypsin deficiency. This substance was then stained by the PAS method (without diastase digestion) and corresponded to amorphous material observed with electron microscopy on adjacent ultrathin sections (Figure 2).
References BUGNON C . , LENYS D. & HERLANT M. (1974) CaractCrisation de diverses cellules de I’adCnohypophyse du Renard par immunofluorescence sur coupes semi-fines et superposition des donnees de microscopie electronique. Comptes Rendus de l’dcademie des Sciences (Paris) 278, I 243-1 247 LANER.P. & EUROPAD.L. (1965) Differential staining of ultrathin sections of epon-embedded tissues for light microscopy. Journal ojHistochemistry and Cytochemistry 13, 579-582 MORIARTY G.C. (1973) Adenohypophysis : Ultrastructural cytochemistry : a review. Journal of Histochemistry und Cytochemistry 21, 855-894 STERNBERGER L.A. (1967) Electron microscopic immunocytochemistry : a review. Journal of Histochemistry and Cytochemistry 15,I 39-1 59 TRAMU G., BEAUVILLAIN J.CL. & DUBOIS M.P. (1974) Les cellules Gonadotropes de l’hypophyse du LBrot (Eliomys quercinus) : concordance des rksultats de microscopie optique (irnmunofluorescence et colorations) et de microscopie Clectronique. Joumai de Microscupie 21, I 8 1-184
