Electron microscopic location of mRNA in the rat kidney: improved post-embedding in situ hybridization.
We determined the optimal technical conditions for post-embedding non-radioactive in situ hybridization applied to ultrastructural location of collagen I mRNA in rat kidney. The signal-to-noise ratio was improved by enhancing hybridization efficiency and distinguishing nonspecific labeling. Probes were labeled with digoxigenin or biotin and detected after hybridization by immunogold or peroxidase techniques. Under these conditions, the signal was located in fibroblasts. With digoxigenin, clusters of gold particles were observed on the endoplasmic reticulum (ER) or scattered throughout the cytoplasmic matrix and nuclei. With the enzymatic method, diaminobenzidine deposits were found on the ER but endogeneous peroxidase partly interfered with the results. Gold particles were less numerous in fibroblast cytoplasm with biotin than with digoxigenin. Moreover, gold particles condensed on fibroblast and tubular cell mitochondria when biotin was used, a phenomenon shown to be due to endogenous biotin by means of a histochemical method. The digoxigenin-immunogold system appeared to be the best method. The biotin system was subject to limitations such as interference from endogenous biotin and poor sensitivity, and mRNA localization was more precise and reliable by the immunogold method than by the enzymatic method.
Yi
J
J
Unité de Recherche Immunopathologie Humaine, INSERM U430, Hôpital Broussais, Paris, France.
Michel
O
O
Sassy-Prigent
C
C
Chevalier
J
J
D000180:Acrylic Resins; D000818:Animals; D001710:Biotin; D003094:Collagen / Q000032:analysis / Q000235:genetics; D004076:Digoxigenin; D017403:In Situ Hybridization / Q000379:methods*; D007668:Kidney / Q000737:chemistry* / Q000648:ultrastructure; D008297:Male; D008854:Microscopy, Electron / Q000379:methods*; D012333:RNA, Messenger / Q000032:analysis*; D051381:Rats; D016707:Tissue Fixation
D000180:Acrylic Resins; D012333:RNA, Messenger; D001710:Biotin; C037883:Lowicryl 4KM resin; D003094:Collagen; D004076:Digoxigenin