HETEROGENEOUS NEGATIVE CHEMOGRAPHY WITH HUMAN ERYTHROCYTES
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PHILIP B. DUNHAM, MARILYN S. KERRAND MARKC. HOROWITZ* Department of Biology, Syracuse University, Syracuse, New York I3210 ABSTRACT.A negative chemographic effect, or fading of the latent image, has been found in autoradiographic studies of human red blood cells. The effect is heterogeneous, being mtricted to a halo-shaped region over each cell.
Chemographic effects are those in which latent images are produced or eliminated in a photographic emulsion by reactive substances in an apposed specimen. Negative chemography, the rapid fading of latent images, has been observed for several kinds of tissues (Boyd 1955, Rogers 1967, Rogers and John 1969). In sections of Submandibular gland from rats, a nonuniform, or heterogeneous negative chemographic effect has been observed; cytoplasm caused more fading of latent images than did nuclei (Rogers and John 1969). This kind of reaction can lead to erroneous conclusions in autoradiographic studies if appropriate controls are not provided. In autoradiographic experiments on unsectioned red blood cells, we have observed a heterogeneous negative chemographic effect which is uniform from cell to cell, but which is restricted to a halo-shaped region over each cell. MATERIALS AND METHODS Human red blood cells, freshly drawn into heparin, were washed three times by centrifugation (4 C, 12,000 X g, 5 min) in an isotonic medium (153 mM NaCI, 17 mM Tris-HC1, 1 mM glucose, pH 7.5). Cells from suspensions of about 30% hematocrit were smeared on microscope slides which had been dipped in a solution of 0.1% gelatin and 0.01% chromium potassium sulfate (subbing medium) and then dried (Caro 1964). The smears were fixed by heating in an oven at 95 C for three hours. Ilford Nuclear Research Emulsion L 4 was diluted I : 1 with distilled water. The smears were dipped in the emulsion, air dried overnight at room temperature, and stored at 4 C. To test for negative chemography, slides prepared in this manner were removed from storage, exposed to room light for 10-20 seconds, and then returned to storage for two weeks. The emulsidns were developed for 4 minutes in Kodak developer D- 19. RFSULTS AND DISCUSSION Figure 1 shows micrographs of portions of a slide prepared to test for negative chemography. The brief exposure of the dried emulsion to light resulted in uniform production of latent images throughout the emulsion since slides developed im'Present address: Department of Pathology, Yale Univenity School of Medicine, New Haven, Conn. 06510
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Biotech Histochem Downloaded from informahealthcare.com by Kungliga Tekniska Hogskolan on 02/04/15 For personal use only.
230
STAIN TECHN0UX;Y
FIG. 1. lmagea of human red blood cells for negative chcmography control prepared as described in the t a t . The bar indicates 5 pn. Main figure is focused on the lower surface of the emulsion; inset is f d on the upper surface.
mediately after exposure to light were uniformly black. During the subsequent storage, latent images were eliminated in specific halo-shaped regions over the cells, and no grains formed in these regions in the lower layer of the emulsion during development. The main portion of Figure 1, which is focused on the lower surface of the emulsion, appears blurred because the grains in the top of the emulsion are out of focus. This point is made clear by the inset, which is focused on the grains in the top of the emulsion. There is little variation in the size and shape of the halos. Their mean outer diameter, 8.8 p, is greater than the mean diameter of the red cells, 7.6 pn, determined on slides prepared in the same manner but not exposed to light. Because of possible spreading of the chemographic effect in the emulsion, the halos may be larger than the region on the cells responsible for the fading of the latent images; the outer rim of the cells may or may not be involved. The mean inner diameter of the halos is 3.5 pm. The significance of this heterogeneous negative chemography is not clear, but those attempting to interpret autoradiographic data from red cells will wish to take it into account. A method is available which may reduce the effect (Sawicki and Danynkiewicz 1963).
NEGATIVE CHEMOGRAPHY WIT H ERYTHROCYTES
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ACKNOWLEDGMENTS We are grateful to Dr. Lowell E. Davis for valuable assistance and advice. This work was supported by USPHS grants NS 08089 and CA 19064.
Biotech Histochem Downloaded from informahealthcare.com by Kungliga Tekniska Hogskolan on 02/04/15 For personal use only.
REFERENCES Boyd, G. A. 1955. Autoradiography in Biology and Medicine. Academic Press, New York, p. 399. Caro, L. G. 1964. High-resolution autoradiography, In: Methods in Cell Physiology, D. M.Prescott, ed. Academic Press, New York pp. 327-363. Rogen, A. W. 1967. Techniques of Autoradiography. Elsevier, Amsterdam. p. 335. Rogers, A. W. and John, P. N. 1969. Latent image stability in autoradiographs of diffusible substances, In: Autoradiography of [email protected] Substances, L. J. Roth a nd W. E. Stumpf, eds. Academic Press,New York. pp. 51-68. Sawicki, W., and Darzynkiewicz, Z. 1963. Polyvinyl chloride protective films in autoradiography. Folia Histochem. Cytochem. I: 283-288.
Biotech Histochem Downloaded from informahealthcare.com by Kungliga Tekniska Hogskolan on 02/04/15 For personal use only.
PHILIP B. DUNHAM, MARILYN S. KERRAND MARKC. HOROWITZ* Department of Biology, Syracuse University, Syracuse, New York I3210 ABSTRACT.A negative chemographic effect, or fading of the latent image, has been found in autoradiographic studies of human red blood cells. The effect is heterogeneous, being mtricted to a halo-shaped region over each cell.
Chemographic effects are those in which latent images are produced or eliminated in a photographic emulsion by reactive substances in an apposed specimen. Negative chemography, the rapid fading of latent images, has been observed for several kinds of tissues (Boyd 1955, Rogers 1967, Rogers and John 1969). In sections of Submandibular gland from rats, a nonuniform, or heterogeneous negative chemographic effect has been observed; cytoplasm caused more fading of latent images than did nuclei (Rogers and John 1969). This kind of reaction can lead to erroneous conclusions in autoradiographic studies if appropriate controls are not provided. In autoradiographic experiments on unsectioned red blood cells, we have observed a heterogeneous negative chemographic effect which is uniform from cell to cell, but which is restricted to a halo-shaped region over each cell. MATERIALS AND METHODS Human red blood cells, freshly drawn into heparin, were washed three times by centrifugation (4 C, 12,000 X g, 5 min) in an isotonic medium (153 mM NaCI, 17 mM Tris-HC1, 1 mM glucose, pH 7.5). Cells from suspensions of about 30% hematocrit were smeared on microscope slides which had been dipped in a solution of 0.1% gelatin and 0.01% chromium potassium sulfate (subbing medium) and then dried (Caro 1964). The smears were fixed by heating in an oven at 95 C for three hours. Ilford Nuclear Research Emulsion L 4 was diluted I : 1 with distilled water. The smears were dipped in the emulsion, air dried overnight at room temperature, and stored at 4 C. To test for negative chemography, slides prepared in this manner were removed from storage, exposed to room light for 10-20 seconds, and then returned to storage for two weeks. The emulsidns were developed for 4 minutes in Kodak developer D- 19. RFSULTS AND DISCUSSION Figure 1 shows micrographs of portions of a slide prepared to test for negative chemography. The brief exposure of the dried emulsion to light resulted in uniform production of latent images throughout the emulsion since slides developed im'Present address: Department of Pathology, Yale Univenity School of Medicine, New Haven, Conn. 06510
229
Biotech Histochem Downloaded from informahealthcare.com by Kungliga Tekniska Hogskolan on 02/04/15 For personal use only.
230
STAIN TECHN0UX;Y
FIG. 1. lmagea of human red blood cells for negative chcmography control prepared as described in the t a t . The bar indicates 5 pn. Main figure is focused on the lower surface of the emulsion; inset is f d on the upper surface.
mediately after exposure to light were uniformly black. During the subsequent storage, latent images were eliminated in specific halo-shaped regions over the cells, and no grains formed in these regions in the lower layer of the emulsion during development. The main portion of Figure 1, which is focused on the lower surface of the emulsion, appears blurred because the grains in the top of the emulsion are out of focus. This point is made clear by the inset, which is focused on the grains in the top of the emulsion. There is little variation in the size and shape of the halos. Their mean outer diameter, 8.8 p, is greater than the mean diameter of the red cells, 7.6 pn, determined on slides prepared in the same manner but not exposed to light. Because of possible spreading of the chemographic effect in the emulsion, the halos may be larger than the region on the cells responsible for the fading of the latent images; the outer rim of the cells may or may not be involved. The mean inner diameter of the halos is 3.5 pm. The significance of this heterogeneous negative chemography is not clear, but those attempting to interpret autoradiographic data from red cells will wish to take it into account. A method is available which may reduce the effect (Sawicki and Danynkiewicz 1963).
NEGATIVE CHEMOGRAPHY WIT H ERYTHROCYTES
231
ACKNOWLEDGMENTS We are grateful to Dr. Lowell E. Davis for valuable assistance and advice. This work was supported by USPHS grants NS 08089 and CA 19064.
Biotech Histochem Downloaded from informahealthcare.com by Kungliga Tekniska Hogskolan on 02/04/15 For personal use only.
REFERENCES Boyd, G. A. 1955. Autoradiography in Biology and Medicine. Academic Press, New York, p. 399. Caro, L. G. 1964. High-resolution autoradiography, In: Methods in Cell Physiology, D. M.Prescott, ed. Academic Press, New York pp. 327-363. Rogen, A. W. 1967. Techniques of Autoradiography. Elsevier, Amsterdam. p. 335. Rogers, A. W. and John, P. N. 1969. Latent image stability in autoradiographs of diffusible substances, In: Autoradiography of [email protected] Substances, L. J. Roth a nd W. E. Stumpf, eds. Academic Press,New York. pp. 51-68. Sawicki, W., and Darzynkiewicz, Z. 1963. Polyvinyl chloride protective films in autoradiography. Folia Histochem. Cytochem. I: 283-288.
