A specific ultrastructural method to reveal DNA: the NAMA-Ur.
We have developed a new, simple, and reproducible cytochemical method to specifically stain DNA at the electron microscopic level: the NAMA-Ur. It is based on the extraction of RNA and phosphate groups from phosphoproteins by a weak alkali hydrolysis (NA) which does not affect DNA, followed by blockage of the amino and carboxyl groups by methylation and acetylation (MA). Finally, sections are stained by uranyl (Ur), which can bind only to DNA. The efficiency of the pre-treatment (NA and MA) was demonstrated by X-ray microanalysis at the transmission electron microscopic level. The NAMA-Ur method has been successfully performed en bloc and on Lowicryl sections in mammalian and plant cells. A specific contrast is observed in the DNA-containing structures after this method, whose sensitivity allows visualization of electron-dense chromatin fibers of 10-12 nm composed of 3-nm DNA fibrils. This staining method has been combined with anti-DNA antibodies, providing complementary information to detect DNA in situ. We propose the NAMA-Ur as an easy method to investigate the chromatin organization in situ at the ultrastructural level.
Testillano
P S
PS
Centro de Investigaciones Biológicas, CSIC, Madrid, Spain.
Sanchez-Pina
M A
MA
Olmedilla
A
A
Ollacarizqueta
M A
MA
Tandler
C J
CJ
Risueño
M C
MC
D000107:Acetylation; D000490:Allium / Q000737:chemistry; D000818:Animals; D002843:Chromatin / Q000737:chemistry / Q000648:ultrastructure; D004247:DNA / Q000032:analysis / Q000648:ultrastructure*; D004577:Electron Probe Microanalysis; D006046:Gold; D006367:HeLa Cells; D006651:Histocytochemistry / Q000379:methods*; D006801:Humans; D006868:Hydrolysis; D008099:Liver / Q000737:chemistry / Q000166:cytology; D008745:Methylation; D051379:Mice; D008854:Microscopy, Electron / Q000379:methods*; D009942:Organometallic Compounds; D011058:Pollen / Q000737:chemistry; D012972:Sodium Hydroxide; D013194:Staining and Labeling / Q000379:methods
D002843:Chromatin; D009942:Organometallic Compounds; C005460:uranyl acetate; D012972:Sodium Hydroxide; D006046:Gold; D004247:DNA