The methylation-acetylation method: an ultrastructural cytochemistry for nucleic acids compatible with immunogold studies.
Methylation-acetylation (MA) is an easy and reproducible ultrastructural cytochemical method which gives preferential contrast to nucleic acid containing structures. When performed en bloc before Lowicryl embedding it does not affect the main antigenic and chemical properties of the sample and is compatible with a large variety of modern immunogold methods permitting a better assignment of the labelling to the well-defined nuclear structures. DNA, RNA, and nuclear proteins, with different chemical nature, nuclear localization, and amount, can be immunolocalized on MA-treated samples. Ultrastructural in situ hybridization and other approaches for studying the functional regions of chromatin, the terminal deoxynucleotidyl transferase and the bromodeoxyuridine methods, are also compatible with the MA procedure. It can be also performed on ultrathin cryosections and Lowicryl sections. A much better visualization of the nuclear structures is obtained, enhancing the distinction between the nucleolar granular and dense fibrillar components. Moreover, the combination of the MA procedure with EDTA regressive staining gives preferential contrast to the RNA-rich structures. It is proposed as a useful approach which can be regularly used for in situ studies of the functional organization of the nucleus in both plant and animal cells.
Testillano
P S
PS
Laboratory of Nuclear Organization during Plant Development, Centro de Investigaciones Biológicas, CSIC, Madrid, Spain.
González-Melendi
P
P
Ahmadian
P
P
Risueño
M C
MC
D000107:Acetylation; D000818:Animals; D002467:Cell Nucleus / Q000737:chemistry / Q000648:ultrastructure; D004247:DNA / Q000032:analysis*; D006651:Histocytochemistry / Q000379:methods*; D007150:Immunohistochemistry; D017403:In Situ Hybridization; D008099:Liver / Q000737:chemistry / Q000648:ultrastructure; D008745:Methylation; D051379:Mice; D010944:Plants / Q000737:chemistry / Q000648:ultrastructure; D012313:RNA / Q000032:analysis*; D013194:Staining and Labeling; D016610:Tissue Embedding
D012313:RNA; D004247:DNA