Microwave-stimulated incubation in immunoelectron microscopy: a quantitative study.
Microwave irradiation has been applied to reduce the immunogold staining time of ultrathin sections of Lowicryl embedded specimens. Labelling has been stimulated by microwave irradiation during incubation with 10 nm colloidal gold particles coated with either goat anti-mouse antibodies (GaM-gold) or goat anti-rabbit antibodies (GaR-gold) and has been compared with control incubations. Quantification has been performed on cytoplasmic membranes or lysosomes labelled with a primary antibody. Counting the gold particles over specific and non-specific sites in electron micrographs and electron microscopic images by IBAS 2000 revealed that irradiation of 25 microliters droplets both at 80 W and 150 W resulted in an accelerated immunogold labelling, while the non-specific background levels were not increased. A plateau level in immunogold labelling intensity was reached after 25 min incubation under microwave irradiation at 150 W as compared to 120 min incubation without microwaves. No improvement in localization sharpness of immunogold labelling on membranes was achieved by microwave irradiation. The microwave-mediated acceleration of immunogold staining may be considered as an example of a staining method with a restricted thermal action on microvolumes as indicated by direct temperature measurements using a fibre-optic thermometer.
Zondervan
P E
PE
Department of Pathology, Erasmus University, Rotterdam, Netherlands.
De Jong
A
A
Sorber
C W
CW
Kok
L P
LP
De Bruijn
W C
WC
Van der Kwast
T H
TH
D000911:Antibodies, Monoclonal; D000954:Antigens, Surface / Q000032:analysis; D006046:Gold; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods; D008247:Lysosomes / Q000648:ultrastructure; D008297:Male; D008654:Mesothelioma / Q000473:pathology / Q000648:ultrastructure*; D008854:Microscopy, Electron / Q000379:methods*; D008872:Microwaves; D011467:Prostate / Q000648:ultrastructure*
D000911:Antibodies, Monoclonal; D000954:Antigens, Surface; D006046:Gold