Dear Colleagues,

I am embarassed to see the interest to my work made so many years ago. I shall scan the reprint and present it here on the next week. In principle the same method I am using in light microscopy of DNA applying instead of uranyl acetate Toluidine blue in half MaIlvain pH 4.0 solution. Very nicely stoichiometric and good contast for morphology, I recommend (the time of hydrolysis may be shortened). As to TEM technique I developed also another method - for negative specific contrast of DNA, using the same depurinising acid hydrolysis with 5N HCl and the  aldehyde blocker phenylhydrazine but likely I have not published it. It is a curious method - phenylhydrazine specifically binds a pseudo-aldehyde group by its hydroxyl and wrapps DNA in benzole hydrophobic coat, which can be negatively stained with tungsten wolframite - the resolution was superior to the positive staining with  Uranylacetate. If anybody would like to play with it - I can try to find the pictures and protocol - it was ages ago. But you should remember that any approach using depurination hydrolysis extracts by it about 30% of euchromatin DNA.

The very high resolution of the chromatin not loosing it can be obtained by the very modern method of localisation microscopy

 http://laser.cheng.cam.ac.uk/wiki/index.php/Localisation_Microscopy    (very time consuming) where you can apply the fluorescent antibodies for histones.

I  would like to use this option and advertise my method of spreading isolated cell nuclei on EM grids (attached).

There much can be gained for studies of the nuclear structure, I also applied here the cytochrome C treatment. Nuclear envelope should be lysed by detergents. Spreading can be so minimal (1-2 min) as to see nearly intact nuclear structure.  Correct preparation of the charged supports is crucial.

Katrina

Hi, there is a method that is called "Ehrenpreisa". Right at the moment, I don't have the reference on the hand. I will give it to you in the next message. Best wishes

Uranyl acetate binds directly to nucleic acids.  If I remember correctly, the stoichiometry is 2:1 phosphate:uranyl.  The staining complex may have more than 1 U atom in it.

Dear Alexandra: I am waiting to your references. thanks a lot

Hi Arash, I send it to you several times, but researchgate was momentaneously out of order. Now, here is the reference:

Erenpreisa J. 1981. Staining of DNA with uranlyacetate in hydrolyzed ultrathin sections. Acta histochem 68(1): 22-26.

I think the reseacher is on researchgate. She is Jekaterina Erenpreisa. I don't have the work. I've only read the reference in a paper of my colleague. Maybe Mrs Jekaterina may send you a copy and even other references and give you some tips. Best wishes.

Arash,

For DNA and DNA plus protein, I usually negative stain with uranyl formate on sample adsorbed carbon grids.  For Chromatin work which needs to be spread on cytochrome C, we use the  high vacuum platinum shadowing technique, both whole mount and replica work.  Hope this helps.

Michael Delannoy

Arash,

guessing you use ultrathin sections applying positive contrast staining: 

Another source (another techniques) - despite from 2002 - could be:

Ribosomal genes in focus: new transcripts label the dense fibrillar components and form clusters indicative of “Christmas trees” in situ.   by:

Karel Koberna ,Jan Malínski , Artem Pliss, Martin Masata , Jaromíra Vecerová , Markéta Fialová , Jan Bednár , and Ivan Raska: in:

JCB vol. 157 no. 5, 743-748

The Rockefeller University Press, doi: 10.1083/jcb.200202007

© 2002 Rockefeller University Press, Published May 28, 2002 

@ http://jcb.rupress.org/content/157/5/743.full.pdf+html

Since you asked about the techniques, this is what I found in my files as the latest:  Using Cryo-Electron Microscopy:

High-resolution structural analysis of a DNA nanostructure by cryoEM,

Takayuki Kato, Russell P. Goodman§, Christoph M. Erben, Andrew J. Turberfield and Keiichi Namba in: Nano Lett., 2009, 9 (7), pp 2747–2750; DOI: 10.1021/nl901265n; Publication Date (Web): June 3, 2009; Copyright © 2009 American Chemical Society; (http://pubs.acs.org/doi/abs/10.1021/nl901265n

Best wishes, Wolfgang

PS: as an "extra" I attach another paper on RNA-TEM (Calabuig et al.2005).

@Arash and @Alexandra:

just to aid with the reference Erenpreisa 1981 see: http://www.sciencedirect.com/science/article/pii/S0065128181800554

The article is availabe only by PPV (pay per view), but as I / one can see from the abstract, ultrathin sections are treated with 5n HCl prior to staining with hydrous UO2Acetate. The article unfortunately is not available via the publication profile of Prof. Dr. Erenpreisa (I asked her today for a setup of the articel in her publication profile but don't know whether she is available or can do us the favour). Nevertheless it seems  necessary to study the whole article to get the idea behind the pretreatment of ultrathin sections (fixation, embedding conditions, grids, etc).

Best wishes and regards, Wolfgang

Dear Colleagues,

I am embarassed to see the interest to my work made so many years ago. I shall scan the reprint and present it here on the next week. In principle the same method I am using in light microscopy of DNA applying instead of uranyl acetate Toluidine blue in half MaIlvain pH 4.0 solution. Very nicely stoichiometric and good contast for morphology, I recommend (the time of hydrolysis may be shortened). As to TEM technique I developed also another method - for negative specific contrast of DNA, using the same depurinising acid hydrolysis with 5N HCl and the  aldehyde blocker phenylhydrazine but likely I have not published it. It is a curious method - phenylhydrazine specifically binds a pseudo-aldehyde group by its hydroxyl and wrapps DNA in benzole hydrophobic coat, which can be negatively stained with tungsten wolframite - the resolution was superior to the positive staining with  Uranylacetate. If anybody would like to play with it - I can try to find the pictures and protocol - it was ages ago. But you should remember that any approach using depurination hydrolysis extracts by it about 30% of euchromatin DNA.

The very high resolution of the chromatin not loosing it can be obtained by the very modern method of localisation microscopy

 http://laser.cheng.cam.ac.uk/wiki/index.php/Localisation_Microscopy    (very time consuming) where you can apply the fluorescent antibodies for histones.

I  would like to use this option and advertise my method of spreading isolated cell nuclei on EM grids (attached).

There much can be gained for studies of the nuclear structure, I also applied here the cytochrome C treatment. Nuclear envelope should be lysed by detergents. Spreading can be so minimal (1-2 min) as to see nearly intact nuclear structure.  Correct preparation of the charged supports is crucial.

Katrina