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Osmium staining of technetium sulfur colloid: A techniqu for electron microscopy
56
Technical Note
Osmium Staining of Technetium Sulfur Colloid : A Technique for Electron Microscopy (Received
26 May 1977)
TECHNIQUES for the intracellular
localization of 99Tcm sulfur colloid and ionic 99Tcm labeled compounds by autoradiography with light microscopy have been previously reported.c’-3’ However, 99Tcm sulfur colloid cannot be visualized in tissue by light microscopy alone or by electron microscopy. Futhermore, autoradiography of 99Tcm sulfur colloid in fixed or frozen tissue sections does not allow adequate localization by electron or light microscopy due to the scatter from 99Tcm beta and gamma radiation.‘4) To obviate these problems, 99Tcm sulfur colloid was prepared using a commercial kit supplied by E. R. Squibb & Sons, New Brunswick, NJ,‘5’in which gelatin was used as a stabilizer.‘@ The kit consists of a 3 ml reaction vial containing 12 mg sodium thiosulfate, 9 mg gelatin, 25.5 mg potassium phosphate and 2.79 mg disodium edetate, a 2 ml syringe containing 0.25 N hydrochloric acid solution, and a 2 ml syringe containing 70mg sodium biphosphate and 20 mg sodium hydroxide. Technetium-99m sulfur colloid (TSC) was prepared by adding 2.0 ml of a sodium pertechnetate solution (99Tcm) from a 99Mo 99Tcm generator to the reaction vial, then adding 2 ml of the 0.25 N hydrochloric acid solution, heating the vial in a boiling water bath for 10min at loo”C, and adding the buffer solution to the cooled vial. Osmium-stained 99Tcm sulfur colloid (OSTSC) was prepared in a similar manner, except that 0.15 ml of osmium tetroxide at a concentration of 1 mg/ml distilled water was added to the reaction vial prior to the addition of the hydrochloric acid solution. Accurate sizing of 99T~m sulfur colloid by conventional electron microscopy exposes the colloid to a high vacuum and may cause the elemental sulfur to sublimate and aggregate. (W TO obviate this problem, a closed wet cell for the electron microscope was utilized to compare 99Tcm sulfur colloid with osmiumstained 99Tcm sulfur colloid!7’ Electron photomicrographs of both the 99Tcm sulfur colloid and the osmium-stained 99Tcm sulfur colloid produced particles of a similar size distribution (Fig. 1). In order to determine biological activity of these two preparations either 300 @Zi 99Tcm sulfur colloid or 300 &i 99Tcm osmium-labeled sulfur colloid was injected intravenously via the tail vein of each of 16 female Sprague-Dawley rats; the animals were sacri-
ficed 30min thereafter and the organ distribution of radioactivity determined. The organ distribution for 99Tcm sulfur colloid and osmium-stained 99Tcm sulfur colloid was not significantly different: liver: 87.5 + 2.3% (&SE.) vs 88.1 i 1.9% (f S.E.); spleen: 1.2 k 0.39~ (&S.E.) vs 1.6 + 0.4% (+S.E.), respectively. Rodent liver sections were prepared according to the following method : 2 ml of the 99Tcm sulfur colloid or osmium-stained 99Tcm sulfur colloid was injected into the tail vein of female Sprague-Dawley rats. The rats were sacrificed 3 hr post injection and the liver removed for fixed sections. A liver sample was placed in a 47/, glutaraldehyde solution in 0.1 M sodium cacodylate buffer at pH 7.4. The sample was then placed in a 0.1 M sodium cacodylate buffer for 3 hr. then fixed in l”/, osmium tetroxide in Millonig’s phosphate buffer at pH 7.4 for 1 hr. The liver sample was dehydrated through graded percentages of ethanol from 20 to 100%. The sample was then infiltrated in a 1 part propylene oxide and 1 part D.E.R. 332-732 plastic mixture overnight. The liver sample is then embedded in the complete plastic mixture and cured 48 hr at 45°C and then 48 hr at 60°C. The standard 99T~m sulfur colloid preparation could not be visualized in liver sections prepared in this manner. The osmium-stained sulfur colloid allowed good visualization of the colloid particles in the rat liver (see Fig. 2). This technique provides a method of visualizing and investigating technetium sulfur colloid localization by means of electron microscopic tissue sections. VA
Hospital and Saint Louis University. St. Louis, MO 63125, U.S.A.
L. R. HENDERSHOTT E. A. GEORGE D. J. KLOS R. M. DONATI
References 1. CHAUDHURI T. K., EVANST. C. and CHAUDHURI T. K. Radiology 109, 633 (1973). 2. QUINONESJ. D. J. nucl. Med. 14, 443 (1973). 3. TILDEN R. L., JACKSON J. JR., ENNEKING W. F., DELAND F. H. and MCVEG J. T. J. nucl. Med. 14, 576 (1973). 4. DEWANJEE M. K. J. nucl. Med. 16, 315 (1975). 5. HANEY T. A.. A.WANIOI. GIGLIOTT~J. A., GusMANOE. A. and BRUNOG. A. J. nucl. Med. 12. 64 (1971). 6. SZYMENDERA J., ZDTOWSKI T., RODWANM. and KORNINSKA J. J. nucl. Med. 12, 212 (1971). 7. FULLMAN E. F. Reu. scient. Instrum. 43, 245 (1972).
sulfur colloid: (a) FIG. I. Electron photomicrographs. 41,500 x . Wet mount of technetium Standard technetium sulfur colloid preparation. (b) Osmium stained technetium sulfur colloid preparation. Note that both preparations show prominent clumping of particles which is not observed in the liver preparation in Fig 2. Comparatively sized particles are indicated by arrows in all preparations demonstrated in Figs. 1 and 2.
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FIG. 2. Electron photomicrograph of rat liver 3 hr after the administration of osmium stained technetium sulfur colloid, 41,500 x , indicates engulfment of sulfur colloid particles by microvilli of sinusoidal liver macrophages (Kupffer cells).
Technical Note
Osmium Staining of Technetium Sulfur Colloid : A Technique for Electron Microscopy (Received
26 May 1977)
TECHNIQUES for the intracellular
localization of 99Tcm sulfur colloid and ionic 99Tcm labeled compounds by autoradiography with light microscopy have been previously reported.c’-3’ However, 99Tcm sulfur colloid cannot be visualized in tissue by light microscopy alone or by electron microscopy. Futhermore, autoradiography of 99Tcm sulfur colloid in fixed or frozen tissue sections does not allow adequate localization by electron or light microscopy due to the scatter from 99Tcm beta and gamma radiation.‘4) To obviate these problems, 99Tcm sulfur colloid was prepared using a commercial kit supplied by E. R. Squibb & Sons, New Brunswick, NJ,‘5’in which gelatin was used as a stabilizer.‘@ The kit consists of a 3 ml reaction vial containing 12 mg sodium thiosulfate, 9 mg gelatin, 25.5 mg potassium phosphate and 2.79 mg disodium edetate, a 2 ml syringe containing 0.25 N hydrochloric acid solution, and a 2 ml syringe containing 70mg sodium biphosphate and 20 mg sodium hydroxide. Technetium-99m sulfur colloid (TSC) was prepared by adding 2.0 ml of a sodium pertechnetate solution (99Tcm) from a 99Mo 99Tcm generator to the reaction vial, then adding 2 ml of the 0.25 N hydrochloric acid solution, heating the vial in a boiling water bath for 10min at loo”C, and adding the buffer solution to the cooled vial. Osmium-stained 99Tcm sulfur colloid (OSTSC) was prepared in a similar manner, except that 0.15 ml of osmium tetroxide at a concentration of 1 mg/ml distilled water was added to the reaction vial prior to the addition of the hydrochloric acid solution. Accurate sizing of 99T~m sulfur colloid by conventional electron microscopy exposes the colloid to a high vacuum and may cause the elemental sulfur to sublimate and aggregate. (W TO obviate this problem, a closed wet cell for the electron microscope was utilized to compare 99Tcm sulfur colloid with osmiumstained 99Tcm sulfur colloid!7’ Electron photomicrographs of both the 99Tcm sulfur colloid and the osmium-stained 99Tcm sulfur colloid produced particles of a similar size distribution (Fig. 1). In order to determine biological activity of these two preparations either 300 @Zi 99Tcm sulfur colloid or 300 &i 99Tcm osmium-labeled sulfur colloid was injected intravenously via the tail vein of each of 16 female Sprague-Dawley rats; the animals were sacri-
ficed 30min thereafter and the organ distribution of radioactivity determined. The organ distribution for 99Tcm sulfur colloid and osmium-stained 99Tcm sulfur colloid was not significantly different: liver: 87.5 + 2.3% (&SE.) vs 88.1 i 1.9% (f S.E.); spleen: 1.2 k 0.39~ (&S.E.) vs 1.6 + 0.4% (+S.E.), respectively. Rodent liver sections were prepared according to the following method : 2 ml of the 99Tcm sulfur colloid or osmium-stained 99Tcm sulfur colloid was injected into the tail vein of female Sprague-Dawley rats. The rats were sacrificed 3 hr post injection and the liver removed for fixed sections. A liver sample was placed in a 47/, glutaraldehyde solution in 0.1 M sodium cacodylate buffer at pH 7.4. The sample was then placed in a 0.1 M sodium cacodylate buffer for 3 hr. then fixed in l”/, osmium tetroxide in Millonig’s phosphate buffer at pH 7.4 for 1 hr. The liver sample was dehydrated through graded percentages of ethanol from 20 to 100%. The sample was then infiltrated in a 1 part propylene oxide and 1 part D.E.R. 332-732 plastic mixture overnight. The liver sample is then embedded in the complete plastic mixture and cured 48 hr at 45°C and then 48 hr at 60°C. The standard 99T~m sulfur colloid preparation could not be visualized in liver sections prepared in this manner. The osmium-stained sulfur colloid allowed good visualization of the colloid particles in the rat liver (see Fig. 2). This technique provides a method of visualizing and investigating technetium sulfur colloid localization by means of electron microscopic tissue sections. VA
Hospital and Saint Louis University. St. Louis, MO 63125, U.S.A.
L. R. HENDERSHOTT E. A. GEORGE D. J. KLOS R. M. DONATI
References 1. CHAUDHURI T. K., EVANST. C. and CHAUDHURI T. K. Radiology 109, 633 (1973). 2. QUINONESJ. D. J. nucl. Med. 14, 443 (1973). 3. TILDEN R. L., JACKSON J. JR., ENNEKING W. F., DELAND F. H. and MCVEG J. T. J. nucl. Med. 14, 576 (1973). 4. DEWANJEE M. K. J. nucl. Med. 16, 315 (1975). 5. HANEY T. A.. A.WANIOI. GIGLIOTT~J. A., GusMANOE. A. and BRUNOG. A. J. nucl. Med. 12. 64 (1971). 6. SZYMENDERA J., ZDTOWSKI T., RODWANM. and KORNINSKA J. J. nucl. Med. 12, 212 (1971). 7. FULLMAN E. F. Reu. scient. Instrum. 43, 245 (1972).
sulfur colloid: (a) FIG. I. Electron photomicrographs. 41,500 x . Wet mount of technetium Standard technetium sulfur colloid preparation. (b) Osmium stained technetium sulfur colloid preparation. Note that both preparations show prominent clumping of particles which is not observed in the liver preparation in Fig 2. Comparatively sized particles are indicated by arrows in all preparations demonstrated in Figs. 1 and 2.
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FIG. 2. Electron photomicrograph of rat liver 3 hr after the administration of osmium stained technetium sulfur colloid, 41,500 x , indicates engulfment of sulfur colloid particles by microvilli of sinusoidal liver macrophages (Kupffer cells).