Novel Non-isotopic Method for the Localization of Receptors in Tissue Sections

Abstract

S U M M A R YWe 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.

Sca-1 + Stem Cell Survival and Engraftment in the Infarcted Heart

“…IGF-1 acts through IGF-1/IGF-1R ligand/receptor system and significantly affects LV remodeling 24. The IGF-1 autocrine and paracrine systems in the viable cardiomyocytes are altered transiently in response to myocardial infarction 25.…”

Dynamic balance of pro‐ and anti‐inflammatory signals controls disease and limits pathology

“…Immunofluorescence techniques identify the spatial organization of cells in granulomas, and then this known organization is combined with flow cytometry data, a simple receptor‐ligand model, and fluorescent microscopy of unbound TNF receptors to reconstruct the relative soluble TNF gradient. A molecular probe from biotinylation of recombinant TNF was constructed to target unbound TNF receptors . In the representative PPD bead granuloma shown, stains are for free TNFR (yellow) using the biotinylated TNF probe and cell nuclei (blue) using DAPI .…”

“…A molecular probe from biotinylation of recombinant TNF was constructed to target unbound TNF receptors. [214][215][216][217][218] In the representative PPD bead granuloma shown, stains are for free TNFR (yellow) using the biotinylated TNF probe and cell nuclei (blue) using DAPI. The dashed white circle indicates approximate PPD bead location.…”

WISP-1 Is an Osteoblastic Regulator Expressed During Skeletal Development and Fracture Repair

“…Binding of WISP-1-Fc to rat embryo sections was evaluated using the in situ ligand-binding procedure previously described. 9,25 No signal was detected when WISP-1-Fc was omitted or the anti-human IgG antibody replaced by an irrelevant antibody (anti-gp 120). The binding pattern described for WISP-1-Fc was unique and different from the binding pattern observed for a control protein (human IgG).…”