[3dem] fixation of dense core granules for immune-EM

Riedel, Dietmar driedel at gwdg.de
Tue Oct 17 06:59:14 PDT 2017

Hi Qiu-Xing,
I agree totally with Paul. HPF is the appropriate Methode to use. Beside I embedded long time ago several pancreas samples using 2.5% glutharaldehyde in 0.1M caccodylic buffer pH 7.1. Fixation was performed at +4*C over night. This worked quite well for pancreas as well as for langerhans islands.
Riedel Dietmar
MPI for biophys. Chemistry

Mobil gesendet

Am 17.10.2017 um 15:36 schrieb Paul Verkade <P.Verkade at bristol.ac.uk<mailto:P.Verkade at bristol.ac.uk>>:

Hi Qiu-Xing,

That is indeed a known problem. Have you tried High Pressure Freezing followed by freeze substitution? This worked very well for us, see attached paper:
Do let me know if you have any questions about the procedure.

Best regards,
Paul Verkade PhD FRMS
Professor of Bioimaging
Wolfson Bioimaging Facility
School of Biochemistry
Biomedical Sciences Building
University of Bristol
University Walk
BS8 1TD Bristol
United Kingdom

Fax. +44-117-3312168
mail: p.verkade at bristol.ac.uk<mailto:p.verkade at bristol.ac.uk>

A top 5 UK university with leading employers (2015)
A top 5 UK university for research (2014 REF)
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On 17 Oct 2017, at 13:19, Jiang,Qiu-Xing <qxjiang at ufl.edu<mailto:qxjiang at ufl.edu>> wrote:

Dear all,

Recently scientists at our facility used a standard fixation/embedding protocol to process insulin-secreting cells in order to preserve the secretory granules (SGs) inside them. Even though most intracellular organelles and structures are pretty well preserved, the preservation of the SG's was very poor and almost all granules were lost, leaving empty space filled with embedding resin. The SG's are known to have very tight membranes, which likely caused the problem. While different protocols are being tested, I am writing to seek some advice from those who might have encountered similar technical issues, and are willing to share their solutions. If so, please email me at qxjiang at ufl.edu<mailto:qxjiang at ufl.edu>. Thanks in advance.


Qiu-Xing Jiang, Ph.D.
Department Microbiology & Cell Science at UF IFAS
Electron Microscopy Faculty Director at UF ICBR
PO Box 110700 | 1355 Museum Drive, Rms 1298 or 1003, Gainesville, FL 32611-0700 USA
Biotech.ufl.edu<https://www.biotech.ufl.edu/> | Tel: 352.846.0953| Fax: 352.392.5933 |E-mail: qxjiang at ufl.edu<mailto:qxjiang at ufl.edu>
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