Visualization and quantitation of peroxisomes using fluorescent nanocrystals: treatment of rats and monkeys with fibrates and detection in the liver.
Peroxisome proliferation in the liver is a well-documented response that occurs in some species upon treatment with hypolipidemic drugs, such as fibrates. Typically, liver peroxisome proliferation has been estimated by direct counting via electron microscopy, as well as by gene expression, enzyme activity, and immunolabeling. We have developed a novel method for the immunofluorescent labeling of peroxisomes, using an antibody to the 70-kDa peroxisomal membrane protein (PMP70) coupled with fluorescent nanocrystals, Quantum Dots. This method is applicable to standard formalin-fixed, paraffin-embedded tissues. Using this technique, a dose-dependent increase in PMP70 labeling was evident in formalin-fixed liver sections from fenofibrate-treated rats. In formalin-fixed liver sections from cynomolgus monkeys given ciprofibrate, quantitative image analysis showed a statistically significant increase in PMP70 labeling compared to control; the increase in hepatic PMP70 protein levels was corroborated by immunoblotting using total liver protein. An increase in hepatic peroxisome number in ciprofibrate-treated monkeys was confirmed by electron microscopy. An advantage of the Quantum Dot/PMP70 method is that a single common protocol can be used to label peroxisomes from several different species, and many of the common problems that arise with immunolabeling, such as fading and low signal strength, are eliminated.
Colton
H M
HM
GlaxoSmithKline, Inc., Safety Assessment, Investigative Toxicology and Pathology, Research Triangle Park, North Carolina 27709, USA. heidi.m.colton@gsk.com
Falls
J G
JG
Ni
H
H
Kwanyuen
P
P
Creech
D
D
McNeil
E
E
Casey
W M
WM
Hamilton
G
G
Cariello
N F
NF
D000818:Animals; D002994:Clofibrate / Q000008:administration & dosage / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D011345:Fenofibrate / Q000008:administration & dosage / Q000494:pharmacology*; D005455:Fluorescent Antibody Technique; D005629:Frozen Sections; D006801:Humans; D015151:Immunoblotting; D008099:Liver / Q000187:drug effects* / Q000378:metabolism / Q000648:ultrastructure; D008252:Macaca fascicularis; D008297:Male; D008565:Membrane Proteins / Q000096:biosynthesis; D008854:Microscopy, Electron; D020675:Peroxisomes / Q000737:chemistry* / Q000378:metabolism; D045663:Quantum Dots; D051381:Rats; D017208:Rats, Wistar; D013045:Species Specificity
D008565:Membrane Proteins; D002994:Clofibrate; D011345:Fenofibrate