Saponin pre-treatment in pre-embedding electron microscopic in situ hybridization for detection of specific RNA sequences in cultured cells: a methodological study.
We describe a method for detection of specific RNA targets in cultured cells at the electron microscopic (EM) level using pre-embedding in situ hybridization (ISH). The specimens were monitored by reflection-contrast microscopy (RCM) before processing for EM. A good balance between preservation of ultrastructure and intensity of hybridization signals was obtained by using mild aldehyde fixation followed by saponin permeabilization. Digoxigenin-labeled probes were used for detection of human elongation factor (HEF) mRNA in HeLa cells, immediate early (IE) mRNA in rat 9G cells, and 28S rRNA in both cell lines. The hybrids were detected immunocytochemically by the peroxidase/diaminobenzidine (DAB) method or by ultra-small gold with silver enhancement. Comparison of these methods favored the peroxidase/DAB system. The accessibility of RNA in the different cell compartments was dependent on the extent of cross-linking during primary fixation even after permeabilization with saponin. By using the most optimal ISH protocol and the peroxidase/DAB system, we detected 28S rRNA over all ribosomes in the cytoplasm but not in the nucleoli, and IE mRNA in a large spot with many smaller spots around it in the nucleoplasm as well as in speckles over the cytoplasm. The sensitivity of the method is such that HEF housekeeping gene transcripts were detected in the cytoplasm.
Macville
M V
MV
Department of Cytochemistry & Cytometry, University of Leiden, The Netherlands.
Wiesmeijer
K C
KC
Dirks
R W
RW
Fransen
J A
JA
Raap
A K
AK
D000818:Animals; D002478:Cells, Cultured; D004076:Digoxigenin; D005347:Fibroblasts / Q000648:ultrastructure; D017781:Genes, Immediate-Early; D006367:HeLa Cells; D006801:Humans; D007124:Immunoenzyme Techniques; D007150:Immunohistochemistry / Q000379:methods; D017403:In Situ Hybridization / Q000379:methods; D007202:Indicators and Reagents; D008854:Microscopy, Electron / Q000379:methods; D010445:Peptide Elongation Factors / Q000096:biosynthesis; D012333:RNA, Messenger / Q000032:analysis*; D012339:RNA, Ribosomal, 28S / Q000032:analysis*; D051381:Rats; D012503:Saponins; D013194:Staining and Labeling / Q000379:methods
D007202:Indicators and Reagents; D010445:Peptide Elongation Factors; D012333:RNA, Messenger; D012339:RNA, Ribosomal, 28S; D012503:Saponins; D004076:Digoxigenin