Abstract
Performing the antibody reaction under controlled heating through microwave irradiation results in significant improvements in the immunovisualization of antigens, such as shortening the times of incubation, lowering the antibody titres used and allowing the detection of difficult, inaccessible antigens. In addition to investigate the basis of the enhancement, we have extended to more intact samples such as isolated plant nuclei and in toto Drosophila embryos the results previously reported only with tissue sections. A drop of heterologous anti-nucleolin antibody covering isolated nuclei from onion root meristems spread on a glass slide was microwave irradiated, resulting in clear immunofluorescent labelling of the nucleoli. This result was never previously obtained in the absence of microwave treatment, even using the complicated procedure previously reported for the homologous identification of this nuclear protein. Using the much larger and to some extent impermeable Drosophila embryos, we were able to show that the incubation time and concentration of the anti-myosin antibody can be strongly reduced by performing the reaction at 45° C under microwave irradiation. The controlled increase in temperature is the main factor responsible for these improvements; the importance of maintaining an adequate mixing of the samples is also emphasized. The proper implementation of these two experimental conditions will require the introduction of appropriate mixing accessories and temperature measuring probes for samples of small volume in current microwave laboratory ovens.
Similar content being viewed by others
References
Ashburner M (1989) Drosophila. A laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York
Bate M (1993) The mesoderm and its derivatives. In: Bate M, Martínez-Arias A (eds) The development of Drosophila melanogaster. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, pp 1013–1090
Boon ME, Hendrikse FC, Kok PG, Bolhuis P, Kok LP (1990) A practical approach to routine immunostaining of paraffin sections in the microwave oven. Histochem J 22: 347–352
Bugler B, Caizergues-Ferrer M, Bouche G, Bourbon HM, Amalric F (1982) Detection and localization of a class of proteins immunologically related to a 100 KDa nucleolar protein. Eur J Biochem 128: 475–480
Campos-Ortega J, Hartenstein V (1989) The embryonic development of Drosophila melanogaster. Springer, Berlin Heidelberg New York
Chilosi M, Mombello A, Montagna L, Benedetti A, Lestani M, Semenzato G, Menestrina F (1990) Multimarker immunohistochemical staining of calgranulins, chloroacetate esterase, and S100 for simultaneous demonstration of inflammatory cells on paraffin sections. J Histochem Cytochem 38: 1669–1675
Chiu KY (1987) Use of microwaves of rapid immunoperoxidase staining of paraffin sections. Med Lab Sci 44: 3–5
Domingo A, Marco R (1989) Visualization under ultraviolet light enhances 100-fold the sensitivity of peroxidase-stained blots. Anal Biochem 182: 176–181
Eisenberg D, Crothers D (1979) Physical chemistry with applications to the life sciences. The Benjamin/Cummings Publishing Company, Menlo Park
Escande ML, Gas N, Stevens B (1985) Immunolocalization of the 100 K nucleolar protein in CHO cells. Biol Cell 53: 99–110
Greenwell A, Foley JF, Maronpot RR (1991) An enhancement method for immunohistochemical staining of proliferating cell nuclear antigen in archival rodent tissues. Cancer Lett 59: 251–256
Hegdé J, Stephenson EC (1993) Distribution of swallow protein in egg chambers and embryos of Drosphila melanogaster. Development 119: 457–470
Hopwood D (1992) Microwaves in tissue preparation. Proc R Microsc Soc 27: 71–74
Kok LP, Boon ME (1990) Microwaves for microscopy. J Microsc 158: 291–322
Lapeyre B, Bourbon HM, Amalric F (1987) Nucleolin, the major nucleolar protein of growing eukaryotic cells: an unusual protein structure revealed by the nucleotide sequence. Proc Natl Acad Sci USA 84: 1472–1476
Leong AS, Milios J (1990) Accelerated immunohistochemical staining by microwaves. J Pathol 161: 327–334
Marco R, Domingo A, Vinós J, Cervera M (1987) In search of the intermediate-like filaments in Drosophila melanogaster. In: Maccioni RB, Arechaga J (eds) The cytoskeleton in cell differentiation and development. Published for International Council of Scientific Unions (ICSU) Press by IRL Press, Oxford Washington, pp 247–256
Martín M, García-Fernández LF, Moreno Díaz de la Espina S, Noaillac-Depeyre J, Gas N, Medina FJ (1992) Identification and localization of a nucleolin homologue in onion nucleoli. Exp Cell Res 99: 74–84
Moreno Díaz de la Espina S, Barthellemy I, Cerezuela MA (1990) Isolation and ultastructural characterization of the residual nuclear matrix in a plant cell system. Chromosoma 100: 110–117
Smith SM (1991) Rapid indirect immunofluorescence of cultured cells facilitated by microwave heating. Bio Techniques 11: 340–342
Sullivan W, Minden JS, Alberts BM (1990) daughterless-abo-like, a Drosophila meternal-effect mutation that exhibits abnormal centrosome separation during the late blastodern divisions. Development 110: 311–323
Wouterlood FG, Boon M, Kok LP (1990) Immunocytochemistry on free-floating sections of rat brain using microwave irradiation during the incubation in the primary antiserum: light and electron microscopy. J Neurosci Methods 35: 133–145
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Medina, F.J., Cerdido, A., Maroto, M. et al. Enhancement of the immunocytochemical detection of antigens by microwave irradiation. Benefits and limitations analysed in isolated plant nuclei and Drosophila embryos in toto. Histochemistry 102, 45–50 (1994). https://doi.org/10.1007/BF00271048
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00271048