, Volume 99, Issue 5, pp 385–390 | Cite as

Selective fluorescence of eosinophilic structures in grasshopper and mammalian testis stained with haematoxylin-eosin

  • J. Espada
  • P. Valverde
  • J. C. Stockert


After staining with Mayer's haematoxylin and eosin Y, paraffin sections of grasshopper and mouse testis were analysed by both transmitted light and fluorescence microscopy. Under violet-blue (436 nm) light excitation, a bright green emission was observed in all eosinophilic structures. Meiotic spindles (fibres and poles), mitochondrial aggregates, centriolar adjuncts in grasshopper spermatids, the basal lamina, flagellar bundles and remaining cytoplasmic droplets in the lumen of seminiferous tubules showed the most striking fluorescence induced by eosin Y. No emission was found in these structures after haemalum staining. Fluorescent microtubular components also revealed a positive immunoperoxidase reaction for α-tubulin. All fixation and embedding procedures (Bouin, Zenker, formaldehyde alone or followed by dichromate or glutaraldehyde, freeze-substitution) were suitable for observation by fluorescence microscopy. Acetylation, deamination, and prolonged washing of stained sections with water, salt solution or ethanol strongly reduced eosin Y fluorescence, while it slightly increased after methylation. These results show that routine haematoxylin-eosin stained tissue sections can be routinely analysed by fluorescence microscopy. The emission of eosin Y allows easy and precise recognition of eosinophilic structures, which are poorly visible under bright field illumination.


Seminiferous Tubule Meiotic Spindle Stain Tissue Section Field Illumination Mammalian Testis 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Armas-Portela R, Stockert JC (1979) Chromatin fluorescence by pyronin staining. Experientia 35:1663–1664Google Scholar
  2. Birkedal-Hansen H (1973) Eosin staining of gelatine. Histochemie 36:73–87Google Scholar
  3. Bobadilla JR, Gutiérrez-Gonzálvez MG, Armas-Portela R, Stockert JC (1988) 9-Fenil-2,3,7-trihidroxi-6-fluorona como colorante y fluorocromo. Acta Bioquim Clin Latinoamer 22:419–425Google Scholar
  4. Burns J, Whitehead R (1966) Staining of Paneth cells with thioflavine T. Nature 221:769–771Google Scholar
  5. Cherry LM, Hsu TC (1984) Antitubulin immunofluorescence studies of spermatogenesis in the mouse. Chromosoma 90:265–274Google Scholar
  6. Cowden RR, Curtis SK (1970) Demonstration of protein-bound sulfhydryl and disulfide groups with fluorescent mercurials. Histochemie 22:247–255Google Scholar
  7. Cowden RR, Curtis SK (1973) Fluorescence cytochemical studies of chromosomes: Quantitative applications of fluorescein mercuric acetate. In: Thaer A, Sernetz M (eds) Fluorescence techniques in cell biology. Springer, Berlin Heidelberg New YorkGoogle Scholar
  8. Del Castillo P, Llorente AR, Stockert JC (1989) Influence of fixation, exciting light and section thickness on the primary fluorescence of samples for microfluorometric analysis. Bas Appl Histochem 33:251–257Google Scholar
  9. Eshaghpour H, Söll D, Crothers DM (1979) Specific chemical labeling of DNA fragments. Nucleic Acids Res 7:1485–1495Google Scholar
  10. Ferrer JM, González-Garrigues M, Stockert JC (1983) Mercurochrome: A fluorescent and electron opaque dye. Microsc Acta 87:293–300Google Scholar
  11. Friedrich K, Seiffert W, Zimmermann HW (1990) Romanowsky dyes and Romanowsky-Giemsa effect. 5. Structural investigations of the purple DNA-AB-EY dye complexes of Romanowsky-Giemsa staining. Histochemistry 93:247–256Google Scholar
  12. Fu CT, Bloch DP (1975) A method for the flow-microfluorometric analysis of histones. Exp Cell Res 93:363–367Google Scholar
  13. Gabe M (1968) Techniques histologiques. Masson, Paris, pp 241–242Google Scholar
  14. Goldstein DJ (1969) The fluorescence of elastic fibres stained with eosin and excited by visible light. Histochem J 1:187–198Google Scholar
  15. Green FJ (1990) The Sigma-Aldrich handbook of stains, dyes and indicators. Aldrich Chemical Company, Milwaukee, WisconsinGoogle Scholar
  16. Grillo TAI, Baxter-Grillo DL (1969) A fluorescent histochemical method for the detection of tissue disulphide groups. Histochemie 18:8–11Google Scholar
  17. Gurr E (1971) Synthetic dyes in biology, medicine and chemistry. Academic Press, London New York, pp 185–200Google Scholar
  18. Hahn von Dorsche H, Opitz M (1970) Beitrag zur Kenntnis der selektiven Fluorochromierung von Mastzellen. Acta Histochem 36:74–86Google Scholar
  19. Hazen MJ, Villanueva A, Juarranz A, Cañete M, Stockert JC (1985) Photosensitizing dyes and fluorochromes as substitutes for 33258 Hoechst in the fluorescence-plus-Giemsa (FPG) chromosome technique. Histochemistry 83:241–244Google Scholar
  20. Horobin RW (1982) Histochemistry. Gustav Fischer, Stuttgart New York, pp 67–68Google Scholar
  21. Knox JP, Dodge AD (1985) The photodynamic action of eosin, a singlet oxygen generator. The inhibition of photosynthetic electron transport. Planta 164:30–34Google Scholar
  22. Lillie RD (1977) H.J. Conn's biological stains, 9th edn. Williams and Wilkins, Baltimore, pp 335–353Google Scholar
  23. Maccioni RB (1986) Molecular cytology of microtubules. Cell Biol Rev 8:3–103Google Scholar
  24. Merck Index (1989) Budavari S (ed) An encyclopaedia of chemicals, drugs and biologicals, 11th edn. Merck & Company, Rahway, New JerseyGoogle Scholar
  25. Pearse AGE (1968) Histochemistry, Theoretical and applied, vol 1, 3rd edn. Churchill Livingstone, Edinburgh London New York, pp 613–619Google Scholar
  26. Rashid F, Horobin RW (1991) Accumulation of fluorescent non-cationic probes in mitochondria of cultured cells: observations, a proposed mechanism, and some implications. J Microsc 163:233–241Google Scholar
  27. Roudier R, Degeyne P (1967) Etude histologique des fibres elastiques par quelques colorants fluorescents. CR Soc Biol 161:2366–2369Google Scholar
  28. Rowe FM (1956–1958; supplement 1963) Colour index, 2nd edn. Bradford, England; Society of Dyers and Colourists, and Lowell, MassachusettsGoogle Scholar
  29. Seliger HH, McElroy WD (1965) Light: physical and biological action. Academic Press, New York London, pp 96, 324–332Google Scholar
  30. Spikes JD (1989) Photosensitization. In: Smith KC (ed) The science of photobiology. Plenum Press, New York, pp 79–110Google Scholar
  31. Stockert JC (1979) Observations on the chromatin staining by aluminium-hematoxylin. Z Naturforsch 34c:1285–1286Google Scholar
  32. Tottrup A, Fredens K, Funch-Jensen P, Aggestrup S, Dahl R (1989) Eosinophil infiltration in primary esophageal achalasia. A possible pathogenic role. Dig Dis Sci 34:1894–1899Google Scholar
  33. Trigoso CI, Del Castillo P, Stockert JC (1992) Influence of inorganic salts on the staining reaction of eosinophil leukocyte granules by anionic dyes. Acta Histochem 93:313–318Google Scholar
  34. Vassar PS, Culling CFA (1959) Fluorescent stains, with special reference to amyloid and connective tissues. Arch Pathol 68:487–498Google Scholar
  35. Waggoner AS (1986) Fluorescent probes for analysis of cell structure, function, and health by flow and imaging cytometry. In: Taylor DL, Waggoner AS, Lanni F, Murphy RF, Birge RR (eds) Applications of fluorescence in the biomedical sciences. Liss, New York, pp 3–28Google Scholar

Copyright information

© Springer-Verlag 1993

Authors and Affiliations

  • J. Espada
    • 1
  • P. Valverde
    • 1
  • J. C. Stockert
    • 1
  1. 1.Departamento de Biología, Facultad de CienciasUniversidad Autónoma de Madrid, CantoblancoMadridSpain

Personalised recommendations