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A method for the stabilization of toluidine blue vital staining for histological and ultrastructural studies
Micron and Microscopica Acta, Vol. 14, No. 4, pp. 353-356, 1983. Printed in Great Britain.
0739 6260/83 $3.00 +0.00 1983 Pergamon Press Ltd.
SHORT COMMUNICATION
A METHOD FOR THE STABILIZATION OF TOLUIDINE BLUE VITAL STAINING FOR HISTOLOGICAL A N D ULTRASTRUCTURAL STUDIES* J. MARNAY AND P. HERLIN Department of Pathology, Centre R6gional Franqois Baclesse, 14021 Caen, France J. H. JACOB Department of Digestive Endoscopy, Centre R6gional Franqois Baclesse, 14021 Caen, France and A. M. MANDARD Department of Pathology, Centre R6gional Franqois Baclesseand Department of Pathology (Electron Microscopy),Centre Hospitalier Universitaire, Caen, France
(Received 23 June 1983)
Abstraet--A method previously developed to stabilize methylene blue vital staining has been applied to toluidine blue, a basic dye with a closely related chemical structure commonly used as a vital stain for the screening of oral and esophageal lesions. The stain was made stable by adding 1% sodium silicotungstate to an aldehyde fixative (formaldehydeor glutaraldehyde). Sections were prepared for light and electron microscopy.
INTRODUCTION It has been found that conventional fixation and embedding techniques when used with toluidine blue do not preserve the staining for subsequent histological studies, due to the high solubility of this dye in aldehyde fixatives and alcohol. Tsuji (1979), Tsuji and Y o k o y a m a (1981), developed a technique for the stabilization of methylene blue, which is a basic dye with a closely related structure. We have adapted this method to preserve toluidine blue staining for both light and electron microscope studies.
* This investigation was supported financially by the 'Ligue Nationale de Lutte contre le Cancer'. 353
MATERIALS AND METHODS A 1~,; solution of toluidine blue (Merck) in distilled water was found to provide optimal results after 5 min staining, followed by extensive rinsing in distilled water. The stain is stabilized by adding 1 ~o sodium silicotungstate (Taab) to the fixative used.
Preparation of material for light microscopy (1) Material was fixed for 18-24 hr in 10~o buffered formaldehyde (Lillie, 1969), to which 1 ~o sodium silicotungstate had been added. (2) Dehydration and embedding was performed as follows: seven changes in absolute methanol, three changes in toluene. One hour for each of the above changes. 58°C
354
J. Marnay, P. Herlin, J. H. Jacob and A. M. Mandard
Fig. 1. Mast cell vital staining with toluidine blue. Glutaraldehyde sodium silicotungstate fixation. Semi-thin section ( x 320}.
Fig. 2. Mast cell vital staining with toluidine blue. Glutaraldehyde sodium silicotungstate fixation. Ultrastructural examination without post-fixation or electron contrast stain. An electron dense precipitate is observed on the granules ( × 15,000t. Insert: detail of granules ( × 30,000t.
Stabilization of Toluidine Blue Vital Staining
Fig. 3. Background contrast introduced by sodium silicotungstate in glutaraldehyde solution. Ultrastructural examination without post-fixation or electron contrast stain ( x 10,000).
Fig. 4. Mast cell. Vital staining with toluidine blue. Glutaraldehyde-sodium silicotungstate fixation. Ultrastructural examination without post-fixation or electron contrast stain. The presence of a sodium silicotungstate crystal near the cell, causing scratches can be seen in the thin section ( x 15,000). Insert: detail of a sodium silicotungstate crystal, observed in the electron microscope ( x 7000).
355
356
J. Marnay, P. Herlin, J. H. Jacob and A. M. Mandard
paraplast, two changes of 90 min each, and then embedded immediately afterwards. (3) Sections of 12-15 tam thickness were cut and studied with optimal nuclear fast red background staining. Preparation of specimens for electron microscopy Fixation was carried out for at least 1 hr in 2~,, glutaraldehyde buffered with sodium cocadylate to which was added 1 ~o sodium silicotungstate. (1) Rinse rapidly in buffer, and dehydrate progressively in ethanol. (2) Substitution in propylene oxide. (3) Infiltration with a mixture of equal parts of propylene oxide and epoxy resin (Maraglas or Epon) (1 hr). (4) Impregnation (overnight) and resin embedding. (5) Semi-thin sections (5-6tam) were examined using optional background staining with a saturated solution of basic fuchsin in distilled water. Thin sections were studied without contrast stain. RESULTS AND DISCUSSION A 1 ~o solution of sodium silicotungstate in an aldehyde fixative has been found to stabilize vital staining with toluidine blue. A purplish blue staining of the areas coloured by the dye was obtained for light microscopy. Deeply stained mast cell granules provide a good example of the results obtained (Fig. 1). The stain is clearly visible in thick sections, and can be combined with a nuclear fast red background stain for paraffin embedded sections, and a basic fuchsin dye for semi-thin sections. The precipitate shows up on non-contrasted ultra-thin sections as electron-dense needles or granules (Fig. 2). While this technique is simple and perfectly reproducible for light microscopy, its use in ultrastructural examination poses certain practical problems.
Osmium tetroxide post-fixation was of no use and should be avoided. The sodium silicotungstate on its own produces a light but adequate contrast to reveal the relevant structures (Fig. 31. The use of osmium tetroxide should be avoided, as it competes with the sodium silicotungstate to form a stained metallic precipitate. The main technical problem resides in the recrystallization of free sodium silicotungstate in the tissue during dehydration with alcohol. The hexagonal silicotungstate crystals (Fig. 4) produced numerous scratches or marks when ultrathin sections were cut using a glass knife. The same crystallization was observed in tissue samples that had not been stained with toluidine blue, but only fixed with sodium silicotungstate (Fig. 3). In conclusion, the method developed by Tsuji to stabilize vital staining of nerve tissue with methylene blue can also be applied to toluidine blue, which is a basic dye of the thiazine group with a closely related chemical structure. This technique should be helpful in understanding the mechanism of toluidine blue in dye tests commonly used for the screening of oral and esophageal lesions.
Acknowledgements -The authors thank Mrs. Blondel, Mr. Bonnet, Mrs. Verbauwhede and Mrs. Mancel for their technical assistance, also Miss Shore for translating the manuscript.
REFERENCES Lillie, R. D., 1969. In: Histochimie Normale et Pathologique. II, Ganter, P. and Jolles, G. (eds.), Gautier Villars, Paris, 1388. Tsuji, S., 1979. Fixation of methylene-blue treated nervous tissue for histological and ultrastructural studies. Naturwissenschaften, 66: 584. Tsuji, S. and Yokoyama, S., 198l. Electron microscopical localization of methyleneblue staining in myentericplexus. Biomed. Res., 2:693 698.
0739 6260/83 $3.00 +0.00 1983 Pergamon Press Ltd.
SHORT COMMUNICATION
A METHOD FOR THE STABILIZATION OF TOLUIDINE BLUE VITAL STAINING FOR HISTOLOGICAL A N D ULTRASTRUCTURAL STUDIES* J. MARNAY AND P. HERLIN Department of Pathology, Centre R6gional Franqois Baclesse, 14021 Caen, France J. H. JACOB Department of Digestive Endoscopy, Centre R6gional Franqois Baclesse, 14021 Caen, France and A. M. MANDARD Department of Pathology, Centre R6gional Franqois Baclesseand Department of Pathology (Electron Microscopy),Centre Hospitalier Universitaire, Caen, France
(Received 23 June 1983)
Abstraet--A method previously developed to stabilize methylene blue vital staining has been applied to toluidine blue, a basic dye with a closely related chemical structure commonly used as a vital stain for the screening of oral and esophageal lesions. The stain was made stable by adding 1% sodium silicotungstate to an aldehyde fixative (formaldehydeor glutaraldehyde). Sections were prepared for light and electron microscopy.
INTRODUCTION It has been found that conventional fixation and embedding techniques when used with toluidine blue do not preserve the staining for subsequent histological studies, due to the high solubility of this dye in aldehyde fixatives and alcohol. Tsuji (1979), Tsuji and Y o k o y a m a (1981), developed a technique for the stabilization of methylene blue, which is a basic dye with a closely related structure. We have adapted this method to preserve toluidine blue staining for both light and electron microscope studies.
* This investigation was supported financially by the 'Ligue Nationale de Lutte contre le Cancer'. 353
MATERIALS AND METHODS A 1~,; solution of toluidine blue (Merck) in distilled water was found to provide optimal results after 5 min staining, followed by extensive rinsing in distilled water. The stain is stabilized by adding 1 ~o sodium silicotungstate (Taab) to the fixative used.
Preparation of material for light microscopy (1) Material was fixed for 18-24 hr in 10~o buffered formaldehyde (Lillie, 1969), to which 1 ~o sodium silicotungstate had been added. (2) Dehydration and embedding was performed as follows: seven changes in absolute methanol, three changes in toluene. One hour for each of the above changes. 58°C
354
J. Marnay, P. Herlin, J. H. Jacob and A. M. Mandard
Fig. 1. Mast cell vital staining with toluidine blue. Glutaraldehyde sodium silicotungstate fixation. Semi-thin section ( x 320}.
Fig. 2. Mast cell vital staining with toluidine blue. Glutaraldehyde sodium silicotungstate fixation. Ultrastructural examination without post-fixation or electron contrast stain. An electron dense precipitate is observed on the granules ( × 15,000t. Insert: detail of granules ( × 30,000t.
Stabilization of Toluidine Blue Vital Staining
Fig. 3. Background contrast introduced by sodium silicotungstate in glutaraldehyde solution. Ultrastructural examination without post-fixation or electron contrast stain ( x 10,000).
Fig. 4. Mast cell. Vital staining with toluidine blue. Glutaraldehyde-sodium silicotungstate fixation. Ultrastructural examination without post-fixation or electron contrast stain. The presence of a sodium silicotungstate crystal near the cell, causing scratches can be seen in the thin section ( x 15,000). Insert: detail of a sodium silicotungstate crystal, observed in the electron microscope ( x 7000).
355
356
J. Marnay, P. Herlin, J. H. Jacob and A. M. Mandard
paraplast, two changes of 90 min each, and then embedded immediately afterwards. (3) Sections of 12-15 tam thickness were cut and studied with optimal nuclear fast red background staining. Preparation of specimens for electron microscopy Fixation was carried out for at least 1 hr in 2~,, glutaraldehyde buffered with sodium cocadylate to which was added 1 ~o sodium silicotungstate. (1) Rinse rapidly in buffer, and dehydrate progressively in ethanol. (2) Substitution in propylene oxide. (3) Infiltration with a mixture of equal parts of propylene oxide and epoxy resin (Maraglas or Epon) (1 hr). (4) Impregnation (overnight) and resin embedding. (5) Semi-thin sections (5-6tam) were examined using optional background staining with a saturated solution of basic fuchsin in distilled water. Thin sections were studied without contrast stain. RESULTS AND DISCUSSION A 1 ~o solution of sodium silicotungstate in an aldehyde fixative has been found to stabilize vital staining with toluidine blue. A purplish blue staining of the areas coloured by the dye was obtained for light microscopy. Deeply stained mast cell granules provide a good example of the results obtained (Fig. 1). The stain is clearly visible in thick sections, and can be combined with a nuclear fast red background stain for paraffin embedded sections, and a basic fuchsin dye for semi-thin sections. The precipitate shows up on non-contrasted ultra-thin sections as electron-dense needles or granules (Fig. 2). While this technique is simple and perfectly reproducible for light microscopy, its use in ultrastructural examination poses certain practical problems.
Osmium tetroxide post-fixation was of no use and should be avoided. The sodium silicotungstate on its own produces a light but adequate contrast to reveal the relevant structures (Fig. 31. The use of osmium tetroxide should be avoided, as it competes with the sodium silicotungstate to form a stained metallic precipitate. The main technical problem resides in the recrystallization of free sodium silicotungstate in the tissue during dehydration with alcohol. The hexagonal silicotungstate crystals (Fig. 4) produced numerous scratches or marks when ultrathin sections were cut using a glass knife. The same crystallization was observed in tissue samples that had not been stained with toluidine blue, but only fixed with sodium silicotungstate (Fig. 3). In conclusion, the method developed by Tsuji to stabilize vital staining of nerve tissue with methylene blue can also be applied to toluidine blue, which is a basic dye of the thiazine group with a closely related chemical structure. This technique should be helpful in understanding the mechanism of toluidine blue in dye tests commonly used for the screening of oral and esophageal lesions.
Acknowledgements -The authors thank Mrs. Blondel, Mr. Bonnet, Mrs. Verbauwhede and Mrs. Mancel for their technical assistance, also Miss Shore for translating the manuscript.
REFERENCES Lillie, R. D., 1969. In: Histochimie Normale et Pathologique. II, Ganter, P. and Jolles, G. (eds.), Gautier Villars, Paris, 1388. Tsuji, S., 1979. Fixation of methylene-blue treated nervous tissue for histological and ultrastructural studies. Naturwissenschaften, 66: 584. Tsuji, S. and Yokoyama, S., 198l. Electron microscopical localization of methyleneblue staining in myentericplexus. Biomed. Res., 2:693 698.