Novel non-isotopic method for the localization of receptors in tissue sections.
We describe a novel fluorescent method for the detection of receptors for chimeric proteins in tissue sections. The technique was developed using a recombinant human insulin-like growth factor (IGF-1) chimera, bearing six additional histidine residues at the carboxy-terminal end (IGF-1-His). We demonstrated that dehydration of the tissue sections was detrimental for binding and that its prevention dramatically increased sensitivity. The specificity of IGF-1-His interaction was shown by gradual abolition of the fluorescent signal in the presence of increasing concentrations of IGF-1. Combining immunofluorescence with in situ ligand binding, we showed that IGF-1-His binding corresponded to the IGF-1 receptor (IGFR-1) distribution in human fetal kidney. Moreover, incubation of the tissue sections with an anti-IGFR-1 blocking antibody abolished IGF-1-His binding, demonstrating that the interaction was mediated by the IGFR-1. The method was also used to localize the IGFR-1 in E18 rat embryo sagittal sections. The IGF-1-His binding pattern was observed in brain, cartilage, lung, skin, heart, diaphragm, and tongue, and paralleled the previously reported IGFR-1 distribution. We believe that this new non-isotopic in situ ligand binding method will facilitate rapid and accurate localization of receptors in tissue sections.
Desnoyers
L
L
Departments of Cell Biology and Technology, Genentech Inc., 1 DNA Way, South San Francisco, California 94080, USA. desnoyer@gene.com
Simonette
R A
RA
Vandlen
R L
RL
Fendly
B M
BM
D000818:Animals; D005333:Fetus; D005455:Fluorescent Antibody Technique; D005629:Frozen Sections; D006639:Histidine / Q000235:genetics; D006801:Humans; D007091:Image Processing, Computer-Assisted; D007334:Insulin-Like Growth Factor I / Q000235:genetics*; D007668:Kidney / Q000378:metabolism; D008024:Ligands; D009928:Organ Specificity; D051381:Rats; D017526:Receptor, IGF Type 1 / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*
D008024:Ligands; D011993:Recombinant Fusion Proteins; D006639:Histidine; D007334:Insulin-Like Growth Factor I; D017526:Receptor, IGF Type 1