A rapid immunogold-silver staining for detection of bromodeoxyuridine in large numbers of plastic sections, using microwave irradiation.
A rapid and convenient method for the large scale, immunogold-silver staining (IGSS) of bromodeoxyuridine (BrdU) incorporated by S phase cells, by means of a monoclonal antibody (anti-BrdU) is described. Nineteen slides at a time can be incubated with the antibodies and the protein A-gold (PAG) in staining jars. The antibody and protein A-gold solutions could be used at least five times to incubate new batches of slides. The incubation times with these solutions were shortened by means of microwave irradiation. In this way 200 slides carrying at least 800 sections could be easily processed under the same conditions in one day, using 1.25 ml neat antibody solutions of anti-BrdU and rabbit anti-mouse. For light microscopy both plastic embedding systems: methylmethacrylate (MMA) and glycolmethacrylate (GMA) can be stained with this technique. The MMA sections, of which the plastic has to be removed before the IGSS, has the advantage of a stronger labelling intensity. The GMA plastic, which contains a cross-linking agent cannot be removed and consequently for GMA sections it is necessary to incubate the sections with a proteolytic enzyme (trypsin) before the IGSS, to reexpose the antigenic binding sides. However, the GMA sections can be allowed to air dry during the IGSS without negative effects on the morphology. This makes it possible to perform the antibody and the PAG-incubating steps on one day and to finish the IGSS the next day. In this way twice as many GMA slides can be incubated with the same antibody and PAG solutions than with MMA slides.(ABSTRACT TRUNCATED AT 250 WORDS)
Department of Cell Biology, State University of Utrecht, Medical School, The Netherlands.
H J
HJ
Van de Kant
A M
AM
Van Pelt
R P
RP
Vergouwen
D G
DG
De Rooij
D000818:Animals; D000911:Antibodies, Monoclonal; D001973:Bromodeoxyuridine / Q000737:chemistry* / Q000276:immunology; D007150:Immunohistochemistry / Q000379:methods*; D008297:Male; D051379:Mice; D008815:Mice, Inbred Strains; D008867:Microtomy; D008872:Microwaves; D010969:Plastics; D013090:Spermatocytes / Q000648:ultrastructure; D013093:Spermatogonia / Q000648:ultrastructure; D013194:Staining and Labeling; D013737:Testis / Q000033:anatomy & histology*
D000911:Antibodies, Monoclonal; D010969:Plastics; D001973:Bromodeoxyuridine