[Use of immunogold labelling technique for immunoelectron microscope localization of proteins in Drosophila polytene chromosomes].
Using gold labeled antibodies, we developed and tested an immunoelectron microscope (IEM) method for detection of protein localization in Drosophila melanogaster polytene chromosomes. This method is based on procedures widely used for indirect immunofluorescent (IF) staining of salivary gland polytene chromosome squashes. The application of IEM was evaluated by using specific antibodies against proteins earlier localized in both decondensed (interbands and puffs) and compact (bands) regions of polytene chromosomes. In all the experiments, IEM and IF images for homologous chromosome regions were compared. When applied to regions of loose structures, IEM enabled us to localize, with high precision, signals in fine bands, interbands and puffs. There was a good correspondence between immunogold EM and IF data. However, there was no correspondence for dense bands: gold particles were distributed at their boundaries, while the entire bands showed bright fluorescence. This discrepancy probably resulted from a poor penetration of antibodies conjugated to gold particles in the tightly packaged structures. From the results obtained it may by concluded that the IEM method is advantageous for studying the fine protein topography of loose decompacted regions of polytene chromosomes. And this must be taken into consideration when protein localization in polytene chromosomes is performed.
Semeshin
V F
VF
Institute of Cytology and Genetics, Siberian Branch of RAS, Novosibirsk 630090, Russia. semenshin@bionet.nsc.ru
Shloma
V V
VV
Andreeva
E N
EN
Saumweber
H
H
Zhimulev
I F
IF
D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry; D029721:Drosophila Proteins / Q000032:analysis*; D004331:Drosophila melanogaster / Q000235:genetics / Q000648:ultrastructure*; D005260:Female; D006046:Gold / Q000737:chemistry*; D007150:Immunohistochemistry; D007814:Larva; D016253:Microscopy, Immunoelectron; D012469:Salivary Glands / Q000648:ultrastructure; D013194:Staining and Labeling; D014960:X Chromosome / Q000648:ultrastructure*
D000911:Antibodies, Monoclonal; D029721:Drosophila Proteins; D006046:Gold