Recuperation and improvement of chromosome preparations stained with silver nitrate

METHODOLOGY

Recuperation and improvement of chromosome preparations stained with silver nitrate

S. Sanchez ^1,3 , J. Cano ² and A.S. Fenocchio ¹

¹Depto. de Genética, Facultad de Ciencias Exactas, Químicas y Naturales, UNaM, Félix de Azara 1552,

3300 Posadas, Misiones, Argentina. Fax: [phone]. Send correspondence to A.S.F.

²Depto. de Biología Celular y Genética, Facultad de Ciencias, Universidad de Málaga, España.

³Depto. de Producción Animal, Facultad de Ciencias Veterinarias, INICNE, SGCyT-UNNE, Sgto. Cabral 2139,

3400 Corrientes, Argentina.

ABSTRACT

Both periodic acid and photographic reagents have been used to remove the silver nitrate residues from cytological preparations. We used potassium ferricyanide to remove AgNO₃ salts in cases of excessive chromosome impregnation. This method produced partial decolorization, with contrast enhancement. Counterstaining with Giemsa also promoted a better contrast between chromosome arms and NORs, which were preserved and retained a dark color. Application of this procedure to chromosome preparations treated sequentially for CBG banding/AgNO₃ staining promoted complete decolorization of C+ regions, leading to "reverse" C-banding, generally with high contrast.

INTRODUCTION

Improvement of chromosome banding techniques has been important for advancement in cytogenetics. In spite of their generalized use, the action of the different chemical agents applied to chromosome preparations is still not clear.

The effects of both C and NOR banding on chromosome preparations seem to be irreversible. However, Dev (1979), using photographic chemical agents and Denton (1989), using periodic acid, have improved cytological preparations treated with argentic impregnation.

MATERIAL AND METHODS

Cytological preparations of human and fish (Lepidosiren paradoxa) cells, obtained with leukocyte culture (Moorhead et al., 1960) and with direct methods (Foresti et al., 1993), were tested. These preparations were stained with silver nitrate, according to the technique described by Howell and Black (1980). For Ag-NOR banding they were then treated with an aqueous K3Fe(CN)6 solution (1% ppv) during a few seconds (1-5 sec), and gently rinsed in water.

These procedures were also applied to previously C-banded chromosome preparations according to Sumner (1972) and sequentially treated with AgNO3. Finally, the material was stained with a Giemsa solution (5%, 2-3 min).

RESULTS AND DISCUSSION

The potassium ferricyanide applied moderately to chromosome preparations, previously stained with AgNO3 caused selective removal of precipitated silver, apparently without any structural chromosome modifications. The chromosome arms progressively lost the brown color but the NORs retained their dark AgNO3 marks. Longer treatments with potassium ferricyanide promoted complete decolorization of the cytological preparations, except for the NORs (Figure 1). These preparations can be stained again, either with AgNO3 or Giemsa, in order to improve the results.

Figure 1 - Human metaphase plates. a, Ag-NOR stained. b, c, The same plate successively destained with K₃Fe(CN)₆. d, Giemsa counterstaining. Note the improvement of the NOR positive dots. The arrows indicate the NORs.

Chromosomes stained successively for both C-banding (CBG) and AgNO3 were also treated with potassium ferricyanide. This procedure promoted differential removal of the characteristic brown gold color from chromosome arms, especially in C bands where all the constitutive heterochromatin "appears" to be removed. In these cases a "reverse" C-banding effect is observed (Figure 2). A peculiar staining pattern resulted with chromosomes in brown, NORs in black and C-positive bands in white, allowing a more accurate determination of C-band location and limits (Figure 2). Counterstaining with Giemsa showed that the heterochromatic regions in these preparations were not significatively affected as they again took on a bright blue color (Figure 3).

Figure 2 - Lepidosiren paradoxa partial metaphase plate. a, Conventional Giemsa staining. b, C-banding. c, "Reverse" C-banding obtained after successive treatments with AgNO₃ and K₃Fe(CN)₆.

Figure 3 - Lepidosiren paradoxa metaphase plate. a, C-banding. b, "Reverse" C-banding (AgNO₃/ K₃Fe(CN)₆ successive treatment). c, Giemsa counterstaining. Note that the C+ bands reacquired coloration.

The lack of effect of K3Fe(CN)6 on the AgNO3-stained chromosome preparations demonstrates the high affinity between silver salts and NOR. In these zones removal of precipitated silver salts was found to be very difficult. This fact could be related to the presence of acid proteins (C23) associated with chromatin (Ochs and Buchs, 1984).

The present method produced fast and easy silver salts removal from AgNO3- and C-banded-treated chromosomes.

ACKNOWLEDGMENTS

The authors are grateful to Dr. L.A.C. Bertollo for critical reading of the manuscript and to Ministerio de Educación y Ciencia de España (Programa de Cooperación con Iberoamérica). Thanks also to Mr. T. Wolkenstein and M.A. Valdéz for collection of the fish used here (Lepidosiren paradoxa) and to G.M. Duarte for translation of the text.

RESUMO

O ácido periódico e reagentes fotográficos têm sido usados para remover resíduos de nitrato de prata de preparações citológicas. Nós usamos ferricianeto de potássio para remover sais de AgNO3 em casos de impregnação cromossômica excessiva. Este método produziu descoloração parcial, acentuando o contraste. Coloração subseqüente com Giemsa também causou um contraste melhor entre os braços cromossômicos e NORs que foram preservados e retiveram uma cor escura. O uso deste procedimento para preparações cromossômicas tratadas seqüencialmente por bandamento CBG e coloração por AgNO3 levou a descoloração global das regiões C+, causando um bandamento C" reverso", geralmente com alto contraste.

(Received July 2, 1996)

• Denton, T.E. (1989). A rapid method for removing silver from staining chromosomes. Stain Technology 64: 49-50.
• Dev, V.G., Miller, D.A., Rechsteiner, M. and Miller, O.J. (1979). Time of suppression of human rRNA gene in mouse-human hybrid cells. Exp. Cell Res. 123: 47-54.
• Foresti, F., Almeida, C. and Foresti de Almeida Toledo, L.A. (1993). A method for chromosome preparations from large fish specimens using in vitro short-term treatment with colchicine. Experientia 49: 810-814.
• Howell, W.M. and Black, D.A. (1980). Controlled silver-staining of nucleolus organizer regions with a protective colloidal developer: a 1-step method. Experientia 36: 1014-1015.
• Moorhead, P.S., Nowell, P.C., Mellman, W.J., Battips, D.M. and Hungerford, D.A. (1960). Chromosome preparations of leukocytes cultured from human peripheral blood. Exp. Cell Res. 20: 613-616.
• Ochs, R.L. and Buchs, H. (1984). Further evidence that phosphoprotein C23 (110 kD/pI 5.1) is the nucleolar silver staining protein. Exp. Cell Res. 152: 260-265.
• Sumner, A.T. (1972). A simple technique for demonstrating centromeric heterochromatin. Expl. Cell Res. 75: 304-306.

Publication Dates

• Publication in this collection
  13 Oct 1998
• Date of issue
  Mar 1997

History

• Received
  02 July 1996