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

Stanislav Vitha stanvitha at tamu.edu
Tue Oct 17 12:29:33 PDT 2017


Sometimes including ~1 %  Acrolein in the fixative can improve fixation (typically 3% formaldehyde, 1-2% glutaraldehyde, 1% Acrolein).  Acrolein penetrates very fast and is a strong crosslinker.  I have also seen DMSO (~5%) included in the fixative to improve penetration.
I have used Malachite Green in fixatives for TEM sample prep for fatty issues; it  is supposed to stain/stabilize lipids.

Using a microwave processor (we have a Pelco Biowave; no commercial interest) for fixation and subsequent steps can also improve the quality of prep.

We have new  HPF and freeze substitution instruments  but so far I have used them for bacteria and yeast only so I cannot provide side by side comparison for other samples.

Stan Vitha
Microscopy and Imaging Center
Texas A&M University
http://microscopy.tamu.edu

From: 3dem [mailto:3dem-bounces at ncmir.ucsd.edu] On Behalf Of Riedel, Dietmar
Sent: Tuesday, October 17, 2017 8:59 AM
To: Paul Verkade
Cc: 3dem at ncmir.ucsd.edu
Subject: Re: [3dem] fixation of dense core granules for immune-EM


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.
Regards
Dietmar
-----------/--
Riedel Dietmar
MPI for biophys. Chemistry
Göttingen

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
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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
-

Qiu-Xing Jiang, Ph.D.
Department Microbiology & Cell Science at UF IFAS
Electron Microscopy Faculty Director at UF ICBR
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