A facile method for immunofluorescence microscopy of highly autofluorescent human retinal sections using nanoparticles with large Stokes shifts.
The human retina is rich in autofluorescent species, such as lipofuscin and melanin. Consequently, it is difficult to localize antigens in the human retina using immunofluorescence microscopy. To address this issue, we have developed a methodology to tag retinal antigens using quantum dot nanoparticles that absorb in the ultraviolet and emit in the infrared, thereby avoiding the visible spectrum. This protocol dramatically improves signal-to-background autofluorescence ratios of immunofluorescence images of human retinal sections, thus enhancing the specific fluorescence in microscopic studies. Of particular note is the ability to detect antigens within the brightly autofluorescent RPE cell layer.
Petty
Howard R
HR
Department of Ophthalmology and Visual Sciences, University of Michigan Medical School, Ann Arbor, MI, United States. hpetty@umich.edu
Elner
Victor M
VM
Kawaji
Takahiro
T
Clark
Andrea
A
Thompson
Debra
D
Yang
Dong-Li
DL
D000368:Aged; D005455:Fluorescent Antibody Technique / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry* / Q000493:pharmacokinetics / Q000528:radiation effects; D006801:Humans; D008027:Light; D008856:Microscopy, Fluorescence / Q000379:methods*; D008875:Middle Aged; D053758:Nanoparticles; D009474:Neurons / Q000166:cytology / Q000502:physiology; D010775:Photic Stimulation / Q000379:methods; D010777:Photochemistry / Q000379:methods; D045663:Quantum Dots; D012160:Retina / Q000737:chemistry / Q000166:cytology*; D013194:Staining and Labeling / Q000379:methods
D005456:Fluorescent Dyes