PMID- 21372809
OWN - NLM
STAT- MEDLINE
DCOM- 20110824
LR  - 20211020
IS  - 1750-2799 (Electronic)
IS  - 1754-2189 (Print)
IS  - 1750-2799 (Linking)
VI  - 6
IP  - 3
DP  - 2011 Mar
TI  - Detection of nascent RNA, single-copy DNA and protein localization by immunoFISH 
      in mouse germ cells and preimplantation embryos.
PG  - 270-84
LID - 10.1038/nprot.2010.195 [doi]
AB  - Dynamic reprogramming of the genome takes place during the gamete-to-embryo 
      transition. This transition defines a period of continuous and global change but 
      has been difficult to study because of extremely limited material and varying 
      degrees of chromatin compaction. Improved methods of detecting chromatin and gene 
      expression changes in the germ line and in the preimplantation embryo would 
      greatly enhance the understanding of this crucial developmental transition. Here 
      we describe a protocol developed and used by us that improves the sensitivity of 
      existing fluorescence in situ hybridization (FISH) methods; the protocol 
      described here has enabled us to visualize single-copy DNA targets and 
      corresponding nascent RNA transcripts in preimplantation embryos and during 
      spermatogenesis. Major improvements over alternative methods involve fixation and 
      permeabilization steps. Chromatin epitopes can be visualized simultaneously by 
      combining FISH with immunofluorescence; multicopy and repetitive element 
      expression can also be reliably measured. This procedure (sample preparation and 
      staining) requires 1-1.5 d to complete and will facilitate detailed examination 
      of spatial relationships between chromatin epitopes, DNA and RNA during the 
      dynamic transition from gamete to embryo.
FAU - Namekawa, Satoshi H
AU  - Namekawa SH
AD  - Department of Molecular Biology, Massachusetts General Hospital, Howard Hughes 
      Medical Institute, Boston, Massachusetts, USA. satoshi.namekawa@cchmc.org
FAU - Lee, Jeannie T
AU  - Lee JT
LA  - eng
GR  - R01 GM058839/GM/NIGMS NIH HHS/United States
GR  - R01 GM098605/GM/NIGMS NIH HHS/United States
GR  - HHMI/Howard Hughes Medical Institute/United States
GR  - R01-GM58839/GM/NIGMS NIH HHS/United States
PT  - Journal Article
PT  - Research Support, N.I.H., Extramural
PT  - Research Support, Non-U.S. Gov't
DEP - 20110210
PL  - England
TA  - Nat Protoc
JT  - Nature protocols
JID - 101284307
RN  - 0 (Chromatin)
RN  - 0 (RNA Precursors)
RN  - 9007-49-2 (DNA)
SB  - IM
MH  - Animals
MH  - Blastocyst/chemistry
MH  - Chromatin/chemistry
MH  - Chromosomes/chemistry
MH  - DNA/*analysis
MH  - Germ Cells/*chemistry
MH  - Immunohistochemistry/*methods
MH  - In Situ Hybridization, Fluorescence/*methods
MH  - Male
MH  - Mice
MH  - RNA Precursors/*analysis
MH  - Repetitive Sequences, Nucleic Acid
MH  - Sensitivity and Specificity
PMC - PMC4335666
MID - NIHMS489813
EDAT- 2011/03/05 06:00
MHDA- 2011/08/25 06:00
PMCR- 2015/02/20
CRDT- 2011/03/05 06:00
PHST- 2011/03/05 06:00 [entrez]
PHST- 2011/03/05 06:00 [pubmed]
PHST- 2011/08/25 06:00 [medline]
PHST- 2015/02/20 00:00 [pmc-release]
AID - nprot.2010.195 [pii]
AID - 10.1038/nprot.2010.195 [doi]
PST - ppublish
SO  - Nat Protoc. 2011 Mar;6(3):270-84. doi: 10.1038/nprot.2010.195. Epub 2011 Feb 10.