Does it need any further preparation, or should we be able to differentiate dense and loose chromatin in TEM? I have stained HeLa cells for a protein which should be associated with euchromatin. I have used the ABC method and now I can't differentiate hetero and euchromatin. I don't see a strong signal anywhere for my protein either.
In my skill, it's quite easy to detect the protein/DNA/RNA localization with Gold via TEM, if you succeeded in detecting that difference with fluorescence staining.
Using standard TEM preparatory techniques, Heterochromatin appears electron-dense and Euchromatin is electron-lucent. This is without any labelling, just heavy-metal staining eg. Uranyl Acetate/ Lead Citrate. Then your labelled protein could be detected against the 'stained' nucleus.
PubMed should provide some direction. Here is just one article.
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3364237/
This is a more expensive path though and it would be simpler if the issues with the inital method could be resolved.
In TEM you can see differences between hetero- and eu-chromation after counterstaining with uranyl acetate and lead citrate. Nucleolar structures for themRNA of ribosomes shows differences of the activation state (see Schwarzacher Hg, 2000)
Hi Pooneh. If you take a look at the article via this link, http://link.springer.com/article/10.1007%2Fs00702-008-0137-1
and look at figure 2, we have ascribed the localisation of our protein of interest in this work to areas of euchromatin; this is based purely on the physical appearance of heterochromatin and euchromatin, as Naomi mentions above. Within this paper there are also details (via links to other papers) of the immunogold labelling TEM approaches we use, which work for the majority of antibodies I've tried. If you need more detail, please mail me on [email protected]
Also, I have used an anti-histone H3 antibody to demark heterochromatin: http://www.lifesci.sussex.ac.uk/home/Julian_Thorpe/TEM95.htm
Good luck, Jules
Dear Pooneh,
just to point you to some (perhaps) interesting articles
(combined LM-EM-methods of diverse kind on Nucleus, Nucleolus, Chromatin):
Since it is not possible to cite those articles in this Forum, I suggest to search PubMed-Search on:
http://www.ncbi.nlm.nih.gov/pubmed/?term=Raska+Ivan
and you will find (at least starting with 2004 up to 2012) a lot of articles of the RASKA-group, most articles have free access, some of them also on/with HeLa cells and most of them contain valued information on techniques. I have not worked in that field, no affiliations with this group, knowing there are a lot of other good sources in the wild, but started from an article I had in my files.
@ Anne JÖRNS:
http://www.ncbi.nlm.nih.gov/pubmed/11173865
Ribosome biogenesis in man: current views on nucleolar structures and function.
Schwarzacher HG, Mosgoeller W.
Cytogenet Cell Genet. 2000;91(1-4):243-52. Review.
@ Pooneh: if you need this article (only PPV, no free access online) please let me know, I do have this one in file.
Good luck, Wolfgang
The best way is to use just standard contrasting procedure with uranyl acetate. In this case heterochromatin in the nucleus appears as more electron dense material than euchromatin.
By standard TEM preparation, using Glutaraldehyde and OsO4 fixation and UrAC and Lead citrate staining, heavy stained heterochromatin and light stained Euchromatin should occur. Only if nucleus is very active, maybe only euchromatin occurs all over.
- Badges
- Science topic
- Similar topics
- Geoscience
- Atmospheric Sciences
- Climate Science
- Climate Change