[3dem] FIB-SEM

Cindi Schwartz cindi.schwartz at Colorado.EDU
Mon Jan 24 13:47:42 PST 2011


Abrahan,

I've done this protocol for 3View--a slice-and-view approach using a diamond knife inside an SEM (so I think the imaging would be very similar). It worked pretty good, but was still a little light on contrast. You might also check your resin. I used the 'hard epon' recipe and maybe something like Spurr's or Durcopan might give better contrast, too.

All fixes and rinses in 0.05M sodium cacodylate buffer

First fix: 2% Gluteraldehyde 30'
Second fix: 2% Osmium tetroxide with 0.8% potassium ferrocyanide 1 hour on ice
Third Fix: 0.15% tannic acid 15'
Fourth Fix: 2% Osmium tetroxide 30'

I think the potassium ferrocyanide will help a lot and that part should be done on ice. The rest I did at room temp.

I took the protocol and modified it from:

Respir Physiol Neurobiol. 2010 Feb 28;170(2):202-9. Epub 2009 Dec 28.
Structure-function studies of blood and air capillaries in chicken lung using 3D electron microscopy.
West JB, Fu Z, Deerinck TJ, Mackey MR, Obayashi JT, Ellisman MH.

I didn't use the "0.1% aqueous thiocarbohydrazide for 20 min" step because it's very toxic and expensive for us to get. It might have made the difference in getting that last bit of contrast, so next time I will.

Good luck!
Cindi

On Jan 24, 2011, at 9:37 AM, Abrahan Hernandez wrote:

> Dear 3dem community, 
> 
> I am performing data acquisition with a FIB-SEM (Focus ion beam- Scanning electron microscope) of metaphase chromosomes in a whole cell. My samples are stained pre-embedding with tannic acid (0.15%, for 1 min), Osmium tetra-oxide (2%, for 1 hour) and uranyl acetate (1%, fir 1 hour). We can see our structures (metaphases chromosomes) but the contrast is not so good, therefore I would like to ask you if you have any recommendation for improve the pre-embedding staining.
> 
> Best
> -----
> Abrahan Hernandez Hernandez, PhD
> Postdoc
> Department of Cell and Molecular Biology (CMB)
> Berzelius Väg 35
> Karolinska Institutet
> S-171 77 Stockholm, Sweden
> Phone: +46 (0)8 5248 7396
> Email: abrahan.hernandez at ki.se
> 
> _______________________________________________
> 3dem mailing list
> 3dem at ncmir.ucsd.edu
> https://mail.ncmir.ucsd.edu/mailman/listinfo/3dem


****************************
Cindi L. Schwartz
University of Colorado
Dept. MCDB
347 UCB
Boulder, CO  80309
O: 303-492-7980
F: 303-735-0770
http://bio3d.colorado.edu
****************************

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