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Detection of different functional states of the cellular nucleus with a new trichrome staining technique
Acta histochem. 71, 73-75 (1982)
Department of Biology, Semmelweis University of Medicine, Budapest
Detection of different functional states of the cellular nucleus with a new trichrome staining technique By
P}~TER
KovAcs, GYORGY CSABA and GY0RGY BALOGHl) With plates II and III (Received .Tanuary 29, 1981)
Summary The new trichrome stain ASTRIN, prepared from alcian blue, safranine, thionine, and resorcin, stains the cell nuclei red or blue, depending on their functional state. Activation of salivary gland cells with pilocarpin, or of liver cell with prednisolone, equally resulted in a quantitative increase of cells with active (blue) nuclei. Changes in nuclear staining after digestion with trypsin, pronase, or ribonuclease, and after extraction of cellular DNA and RNA with perchloric acid, unequivocally suggest that alteration of the protein-DNA ratio, i.e. the hetero- or euchromatic state of nuclear chromatin, is responsible for the disparity of the nuclear staining reaction with ASTRIN. The new trichrome stain seems to be suitable for routine cytodiagnostic use.
Introduction The new trichrome stain ASTRIN, described by us in a previous paper (BALOGH et al. 1981), was prepared from alcian blue, safranine, thionine, and resorcin. It proved to be suitable for the differential staining of tissues and of cell nuclei in different functional states. In preliminary examinations, the nuclear chromatin stained blue with ASTRIN in the heterochromatic state and red in the euchromatic state. This prompted closer examinations on the value of ASTRIN in detecting nuclear activity, for only few simple techniques are available for differential staining of nuclei in different functional states.
Material and Method 1. Liver and pancreas of adult rats were fixed in Carnoy's solution, embedded in Histoplast, and cut to sections which were processed as follows: a) Digestion with 0.25 % trypsin (Dileo, Michigan) for 5 min, 2 washes in PBS (0.05 rn phosphate buffer, pH = 7.2 0.9% NaCI), staining with ASTRIN. b) Digestion with 0.25 % pronase (Calb1:ochem, USA, B-grade) for 1 Ii, 2 washes in PBS, staining with ASTRIN. c) Digestion with 0.1 % ribonuclease (Reanal, Budapest) solution in 0.2 M veronal-HCI buffer (pH = 7.7) for I h (PEARSE 1972), 2 washes in PBS, staining with ASTRIN.
+
1) Present address: Hungarocamion, Budapest.
74
P. KovAc s, G. CSABA and G.
BALOGH
d) Ext.-action of RNA a nd DNA with 5 0/., perchloric acid for 30 min, neutralization in 1 % NaCHO a solution, rinsing in tap water (CHAYEN et al. 1973), and s taining with ASTRIN. 2. Male rats 100 g in we ight were treated with 0.3 mg pilocarpin by intraperitoneal route. 5 min later the submandibular salivary gland was removed, fixed in Carnoy's solution, embedded in Histopla st, and stained with ASTRIN. 3. Male rats weighing 100 g were tre ated intraperitoneally with 5 mg prednisolon (Di-Adreson Fra quosUID; Organon-Oss, Holla nd). 1 h later liver biopsies were t ak en, fixed in Carnoy's solution, e mbedded in Hislop/as!, cut to sec tions, and stained with ASTRIN_ 4. Crock er 180 mouse sarcoma was fixed in Carnoy's solution, a nd wa s stained with ASTRIN a fter histologi cal process ing. 5. Tiss ues of animals at low levels of phylogenetic or ontoge ne ti c d e velopment (hydra and frog la rva, res pec tively), and testi cul a r ti ssue of adult rats were sta ined swith ASTRIN after histological process ing.
Results and Discussion Binding of red stain was considerably decreased in both liver and pancreas preparations after digestion with trypsin (Fig. 1, plate II). Only the areas corresponding to nucleoli, certain highly condensed segments of chromatin, and the chromosomes of dividing cells, stained red with ASTRIN , while in the control preparations the cell nuclei alternately took on blue and red stain (Fig. 2, plate II). Digestion with pronase had similar results, except that the ergastoplasmic islets preserved their reddish-violet shade (Fig . 3, plate II). Digestion with ribonuclease accounted for increase in the number of red-staining nuclei (Fig. 4, plate III). Since both trypsin and pronase digest proteins - at different intensity - they presumably removed the proteins of chromatin - histones and nonhistones - from the nucleus. This does not indicate the binding of safranine to the proteins, but suggests the responsibility of the configuration developed by proteins - degree of spira liz at ion - for staining with safranine, i.e. for metachromasia. This is also supported by the observation that red-staining nuclei disappeared after extraction with perchloric acid to the same extent as after trypsin or pronase digestion. It follows that the sta.ining reaction depends not so much on binding to a specific molecule as on alteration of the quantitative relation between all components involved. The fact that the heads of all spermatozoa (in testicular tissue) took on red stain, accords well with this implication, for the sperms, being in a state of maximal information block, are extraordinarily heterochromatic. The non-uniform functional alteration of the nuclei after the different digestion procedures remains to be explained; at all events, it suggets the possible existence of a digestion-resistant state of chromatin condensation. Pilocarpin is known to activate the salivary gland cells, which show a marked secretory activity under its influence. Staining with ASTRIN revealed increase of active (blue) nuclei to 77.6 % from 55.8 % found without pilocarpin treatment. Prednisolone, known as gene activating glycocorticoid, had a similar effect: the proportion of blue nuclei increased from 62.9 to 7l.9 % in a relatively short time. The Crocker mouse sarcoma cells (Fig. 5, plate III) has a loose structure. Their thin chromatin network stained blue and their nucleoli, as well as the chromosomes of dividing cells, stained red. The nuclei of differentiating cell populations (of the frog
Acta histochem. Vol. 71
Plate II
1
2
3 Kovacs, Csa ba and Balogh VEB GUSTAV FISCHER VERLAG lENA
A eta histoehem. Vol. 71
Plate III
4
Kovacs, Csaba and Balogh VEB GUSTAV FISCHER VERLAG JENA
Detection of different functional states of the cellular nucleus
75
larva) also stained blue. Such cells are inhibited by proteins to a lesser degree than are mature (normal) cells. Most nuclei stained blue in organisms representing a low level of phylogenesis, at which the amount of cellular DNA, and hence blocking, is lower than e.g. at the level of the differentiated mammalian cell. The experiments have unequivocally shown that the active (activated) cellular nuclei stain blue with ASTRIN, while inactive - functionally inhibited - nuclei stain red with it. The mechanism responsible for the staining reaction presumably depends on the quantitative relation between protein and DNA, i.e. on the proteininduced changes of the DNA configuration. The ASTRIN staining technique thus seems to be suitable for detection of the active or inactive state of nuclei, and might probably be utilized for the functional cytological diagnosis of tumour cells.
I,iterature BALOGH, G., KovAcs, P., and CSABA, G., ASTRIN - a new trichome straining technique. Z. mikrosk.·anat. Forse-h. 96, 138-144 (1982). Under publication (1981). CHAYEN, .J., BITENSKY, K., and BUTCHER, R., Practical histochemistry. \Viley, London, New York 1973. PEARSE, A. G. E., Histochemistry. Theoretical and applied. 2nd ed. Chmchill, Livingstone; Edinburgh and London 1972. Address: Prof. Dr. G. CSABA, Department of Biology, Semmelweis University of Medicine, Nagyvarad tel'. 4. POB 370. H - 1445 Budapest.
Explanation to plates II and III Fig. 1. Rat liver stained with ASTRIN after digestion with trypsin. Most nuclei are blue, but all nucleoli are red. X 1,200. Fig. 2. Section f!'Om normal liver (contr·ol). Roughly equal proportions of the nucleoli took on blue and reel stain. All nucleoli are red. X 1,200. Fig. 3. Rat liver after digestion with pronase. Most nucleoli are blne, the cytoplasm is generaJly reddish-violet. X 1,200. Fig. 4. Rat liver after digestion with RNAse. Most nucleoli are red. X 1,200. Fig. 5. Crocker S 180 mouse sarcoma cells stained with ASTRIN. The nuclear structure is loose; only the nncleoli stain red, the chromatin stains blue. Note anaphasic cell with vivid red chromosomes. X 1,200.
Department of Biology, Semmelweis University of Medicine, Budapest
Detection of different functional states of the cellular nucleus with a new trichrome staining technique By
P}~TER
KovAcs, GYORGY CSABA and GY0RGY BALOGHl) With plates II and III (Received .Tanuary 29, 1981)
Summary The new trichrome stain ASTRIN, prepared from alcian blue, safranine, thionine, and resorcin, stains the cell nuclei red or blue, depending on their functional state. Activation of salivary gland cells with pilocarpin, or of liver cell with prednisolone, equally resulted in a quantitative increase of cells with active (blue) nuclei. Changes in nuclear staining after digestion with trypsin, pronase, or ribonuclease, and after extraction of cellular DNA and RNA with perchloric acid, unequivocally suggest that alteration of the protein-DNA ratio, i.e. the hetero- or euchromatic state of nuclear chromatin, is responsible for the disparity of the nuclear staining reaction with ASTRIN. The new trichrome stain seems to be suitable for routine cytodiagnostic use.
Introduction The new trichrome stain ASTRIN, described by us in a previous paper (BALOGH et al. 1981), was prepared from alcian blue, safranine, thionine, and resorcin. It proved to be suitable for the differential staining of tissues and of cell nuclei in different functional states. In preliminary examinations, the nuclear chromatin stained blue with ASTRIN in the heterochromatic state and red in the euchromatic state. This prompted closer examinations on the value of ASTRIN in detecting nuclear activity, for only few simple techniques are available for differential staining of nuclei in different functional states.
Material and Method 1. Liver and pancreas of adult rats were fixed in Carnoy's solution, embedded in Histoplast, and cut to sections which were processed as follows: a) Digestion with 0.25 % trypsin (Dileo, Michigan) for 5 min, 2 washes in PBS (0.05 rn phosphate buffer, pH = 7.2 0.9% NaCI), staining with ASTRIN. b) Digestion with 0.25 % pronase (Calb1:ochem, USA, B-grade) for 1 Ii, 2 washes in PBS, staining with ASTRIN. c) Digestion with 0.1 % ribonuclease (Reanal, Budapest) solution in 0.2 M veronal-HCI buffer (pH = 7.7) for I h (PEARSE 1972), 2 washes in PBS, staining with ASTRIN.
+
1) Present address: Hungarocamion, Budapest.
74
P. KovAc s, G. CSABA and G.
BALOGH
d) Ext.-action of RNA a nd DNA with 5 0/., perchloric acid for 30 min, neutralization in 1 % NaCHO a solution, rinsing in tap water (CHAYEN et al. 1973), and s taining with ASTRIN. 2. Male rats 100 g in we ight were treated with 0.3 mg pilocarpin by intraperitoneal route. 5 min later the submandibular salivary gland was removed, fixed in Carnoy's solution, embedded in Histopla st, and stained with ASTRIN. 3. Male rats weighing 100 g were tre ated intraperitoneally with 5 mg prednisolon (Di-Adreson Fra quosUID; Organon-Oss, Holla nd). 1 h later liver biopsies were t ak en, fixed in Carnoy's solution, e mbedded in Hislop/as!, cut to sec tions, and stained with ASTRIN_ 4. Crock er 180 mouse sarcoma was fixed in Carnoy's solution, a nd wa s stained with ASTRIN a fter histologi cal process ing. 5. Tiss ues of animals at low levels of phylogenetic or ontoge ne ti c d e velopment (hydra and frog la rva, res pec tively), and testi cul a r ti ssue of adult rats were sta ined swith ASTRIN after histological process ing.
Results and Discussion Binding of red stain was considerably decreased in both liver and pancreas preparations after digestion with trypsin (Fig. 1, plate II). Only the areas corresponding to nucleoli, certain highly condensed segments of chromatin, and the chromosomes of dividing cells, stained red with ASTRIN , while in the control preparations the cell nuclei alternately took on blue and red stain (Fig. 2, plate II). Digestion with pronase had similar results, except that the ergastoplasmic islets preserved their reddish-violet shade (Fig . 3, plate II). Digestion with ribonuclease accounted for increase in the number of red-staining nuclei (Fig. 4, plate III). Since both trypsin and pronase digest proteins - at different intensity - they presumably removed the proteins of chromatin - histones and nonhistones - from the nucleus. This does not indicate the binding of safranine to the proteins, but suggests the responsibility of the configuration developed by proteins - degree of spira liz at ion - for staining with safranine, i.e. for metachromasia. This is also supported by the observation that red-staining nuclei disappeared after extraction with perchloric acid to the same extent as after trypsin or pronase digestion. It follows that the sta.ining reaction depends not so much on binding to a specific molecule as on alteration of the quantitative relation between all components involved. The fact that the heads of all spermatozoa (in testicular tissue) took on red stain, accords well with this implication, for the sperms, being in a state of maximal information block, are extraordinarily heterochromatic. The non-uniform functional alteration of the nuclei after the different digestion procedures remains to be explained; at all events, it suggets the possible existence of a digestion-resistant state of chromatin condensation. Pilocarpin is known to activate the salivary gland cells, which show a marked secretory activity under its influence. Staining with ASTRIN revealed increase of active (blue) nuclei to 77.6 % from 55.8 % found without pilocarpin treatment. Prednisolone, known as gene activating glycocorticoid, had a similar effect: the proportion of blue nuclei increased from 62.9 to 7l.9 % in a relatively short time. The Crocker mouse sarcoma cells (Fig. 5, plate III) has a loose structure. Their thin chromatin network stained blue and their nucleoli, as well as the chromosomes of dividing cells, stained red. The nuclei of differentiating cell populations (of the frog
Acta histochem. Vol. 71
Plate II
1
2
3 Kovacs, Csa ba and Balogh VEB GUSTAV FISCHER VERLAG lENA
A eta histoehem. Vol. 71
Plate III
4
Kovacs, Csaba and Balogh VEB GUSTAV FISCHER VERLAG JENA
Detection of different functional states of the cellular nucleus
75
larva) also stained blue. Such cells are inhibited by proteins to a lesser degree than are mature (normal) cells. Most nuclei stained blue in organisms representing a low level of phylogenesis, at which the amount of cellular DNA, and hence blocking, is lower than e.g. at the level of the differentiated mammalian cell. The experiments have unequivocally shown that the active (activated) cellular nuclei stain blue with ASTRIN, while inactive - functionally inhibited - nuclei stain red with it. The mechanism responsible for the staining reaction presumably depends on the quantitative relation between protein and DNA, i.e. on the proteininduced changes of the DNA configuration. The ASTRIN staining technique thus seems to be suitable for detection of the active or inactive state of nuclei, and might probably be utilized for the functional cytological diagnosis of tumour cells.
I,iterature BALOGH, G., KovAcs, P., and CSABA, G., ASTRIN - a new trichome straining technique. Z. mikrosk.·anat. Forse-h. 96, 138-144 (1982). Under publication (1981). CHAYEN, .J., BITENSKY, K., and BUTCHER, R., Practical histochemistry. \Viley, London, New York 1973. PEARSE, A. G. E., Histochemistry. Theoretical and applied. 2nd ed. Chmchill, Livingstone; Edinburgh and London 1972. Address: Prof. Dr. G. CSABA, Department of Biology, Semmelweis University of Medicine, Nagyvarad tel'. 4. POB 370. H - 1445 Budapest.
Explanation to plates II and III Fig. 1. Rat liver stained with ASTRIN after digestion with trypsin. Most nuclei are blue, but all nucleoli are red. X 1,200. Fig. 2. Section f!'Om normal liver (contr·ol). Roughly equal proportions of the nucleoli took on blue and reel stain. All nucleoli are red. X 1,200. Fig. 3. Rat liver after digestion with pronase. Most nucleoli are blne, the cytoplasm is generaJly reddish-violet. X 1,200. Fig. 4. Rat liver after digestion with RNAse. Most nucleoli are red. X 1,200. Fig. 5. Crocker S 180 mouse sarcoma cells stained with ASTRIN. The nuclear structure is loose; only the nncleoli stain red, the chromatin stains blue. Note anaphasic cell with vivid red chromosomes. X 1,200.