Biocompatible fluorescent nanocrystals for immunolabeling of membrane proteins and cells.
A methodology for simple convenient preparation of bright, negatively or positively charged, water-soluble CdSe/ZnS core/shell nanocrystals (NCs) and their stabilization in aqueous solution is described. Single NCs can be detected using a standard epifluorescent microscope, ensuring a detection limit of one molecule coupled with an NC. NCs solubilized in water by DL-Cys were stabilized, to avoid aggregation, by poly(allylamine) and conjugated with polyclonal anti-mouse antibodies (Abs). NC-Abs conjugates were tested in dot-blots and exhibited retention of binding capacity within several nanograms of antigen detected. We further demonstrated the advantages of NC-Abs conjugates in the immunofluorescent detection and three-dimensional (3D) confocal analysis of p-glycoprotein (p-gp), one of the main mediators of the MDR phenotype, overexpressed in the membrane of MCF7r breast adenocarcinoma cells. Immunolabeling of p-gp with NC-Abs conjugates was 4200-, 2600-, and 420-fold more resistant to photobleaching than its labeling with fluorescein isothiocyanate-Abs, R-phycoerythrin-Abs, and AlexaFluor488-Abs, respectively. The labeling of p-gp with NC-Abs conjugates was highly specific, and the data were used for confocal reconstruction of 3D images of the p-gp distribution in the MCF7r cell membrane. Finally, we demonstrated the applicability of NC-Abs conjugates obtained by the method described to specific detection of antigens in paraffin-embedded formaldehyde-fixed cancer tissue specimens, using immunostaining of cytokeratin in skin basal carcinoma as an example. We conclude that the NC-Abs conjugates may serve as easy-to-do, highly sensitive, photostable labels for immunofluorescent analysis, immunohistochemical detection, and 3D confocal studies of membrane proteins and cells.
Sukhanova
Alyona
A
Institut Fédératif de Recherche No. 53 Biomolécules, Université de Reims Champagne-Ardenne, 51100 Reims, France.
Devy
Jérôme
J
Venteo
Lydie
L
Kaplan
Hervé
H
Artemyev
Mikhail
M
Oleinikov
Vladimir
V
Klinov
Dmitry
D
Pluot
Michel
M
Cohen
Jacques H M
JH
Nabiev
Igor
I
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000032:analysis* / Q000737:chemistry; D000906:Antibodies / Q000737:chemistry / Q000276:immunology; D002462:Cell Membrane / Q000737:chemistry*; D016650:Fluorescein-5-isothiocyanate; D005455:Fluorescent Antibody Technique / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry*; D006801:Humans; D007633:Keratins / Q000032:analysis / Q000737:chemistry; D008565:Membrane Proteins / Q000032:analysis* / Q000737:chemistry / Q000276:immunology; D018613:Microscopy, Confocal / Q000379:methods; D008854:Microscopy, Electron / Q000379:methods; D036103:Nanotechnology; D011073:Polyamines; D011806:Quinolinium Compounds; D018036:Selenium Compounds / Q000737:chemistry / Q000276:immunology; D013440:Sulfides / Q000737:chemistry / Q000276:immunology; D014407:Tumor Cells, Cultured; D017967:Zinc Compounds / Q000737:chemistry / Q000276:immunology
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; C481052:Alexa fluor 546; D000906:Antibodies; D005456:Fluorescent Dyes; D008565:Membrane Proteins; D011073:Polyamines; D011806:Quinolinium Compounds; D018036:Selenium Compounds; D013440:Sulfides; D017967:Zinc Compounds; C063994:polyallylamine; D007633:Keratins; D016650:Fluorescein-5-isothiocyanate; C031238:zinc sulfide