ISH-IEM: a sensitive method to detect endogenous mRNAs at the ultrastructural level.
This protocol describes the combination of in situ hybridization (ISH) with cryo-immunolabeling methods to allow the simultaneous detection at the ultrastructural level of mRNAs and proteins. The procedure consists of five steps and takes 4-5 d: (i) acquisition of ultrathin frozen sections of chemically fixed tissues or cells; (ii) hybridization of the sections with digoxigenin (DIG) or biotin-labeled RNA probes; (iii) detection of the bound probe with antibodies and protein A-gold (PAG); (iv) labeling of proteins of interest (optional); and (v) visualization by transmission electron microscopy (immuno-electron microscopy (IEM)). This technique allows the simultaneous detection of endogenous/overexpressed/injected RNAs and proteins while preserving the cell ultrastructure. The protocol is also suitable for mRNA detection on semi-thin frozen sections in combination with immunofluorescence. The localization of targeted transcripts, such as gurken and oskar mRNA in the Drosophila oocyte, and of structural elements and proteins that mediate their localization have been revealed using this technique.
Herpers
Bram
B
Department of Cell Biology, Cell Microscopy Centre, UMC Utrecht, Heidelberglaan, Utrecht, The Netherlands.
Xanthakis
Despina
D
Rabouille
Catherine
C
D000818:Animals; D004330:Drosophila / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure; D029721:Drosophila Proteins / Q000235:genetics; D005260:Female; D005629:Frozen Sections; D017344:Genes, Insect; D017403:In Situ Hybridization / Q000379:methods*; D016253:Microscopy, Immunoelectron / Q000379:methods*; D009865:Oocytes / Q000378:metabolism / Q000648:ultrastructure; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D016211:Transforming Growth Factor alpha / Q000235:genetics
D029721:Drosophila Proteins; D012333:RNA, Messenger; D016211:Transforming Growth Factor alpha; C083730:grk protein, Drosophila; C091675:osk protein, Drosophila