Enhancement of the immunocytochemical detection of antigens by microwave irradiation. Benefits and limitations analysed in isolated plant nuclei and Drosophila embryos in toto.
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 degrees 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.
Medina
F J
FJ
Centro de Investigaciones Biológicas (CSIC), Madrid, Spain.
Cerdido
A
A
Maroto
M
M
Manzanares
M
M
Marco
R
R
D000818:Animals; D000937:Antigen-Antibody Reactions; D000941:Antigens / Q000032:analysis*; D004330:Drosophila / Q000737:chemistry* / Q000196:embryology*; D004625:Embryo, Nonmammalian / Q000528:radiation effects; D005455:Fluorescent Antibody Technique; D007150:Immunohistochemistry; D008872:Microwaves; D009218:Myosins / Q000032:analysis*; D009687:Nuclear Proteins / Q000032:analysis*; D009697:Nucleolus Organizer Region / Q000737:chemistry; D010750:Phosphoproteins / Q000032:analysis*; D010940:Plant Proteins / Q000032:analysis*; D018517:Plant Roots / Q000737:chemistry; D010944:Plants / Q000737:chemistry* / Q000528:radiation effects; D016601:RNA-Binding Proteins; D000097796:Nucleolin
D000941:Antigens; D009218:Myosins; D009687:Nuclear Proteins; D010750:Phosphoproteins; D010940:Plant Proteins; D016601:RNA-Binding Proteins; D000097796:Nucleolin