[Histonet] RE: RE: Specimens for TEM

[Sherwood, Margaret]

Just one minor change in the below mentioned protocol.  Do you really do "water" washes?  I have never heard of that.  We do buffer rinses.  (I personally used Cacodylate buffer, but people also use Phosphate buffer).

 


Peggy Sherwood
Research Specialist, Photopathology
Wellman Center for Photomedicine (EDR 214)
Massachusetts General Hospital
50 Blossom Street
Boston, MA 02114-2696
617-724-4839 (voice mail)
617-726-6983 (lab)
617-726-1206 (fax)
msherwood <@t> partners.org

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Duraine, Lita
Sent: Friday, October 05, 2012 1:22 PM
To: Histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] RE: Specimens for TEM

Hi Allyse,
TEM is a whole new ball of wax so to speak.  It depends on what you are fixing, and how you plan to fix it.  Typically you would start with Paraformaldehyde and a Glutaraldehyde mix such as Karnovsky's.  The after water washes do a secondary fix in Osmium Tetroxide followed by water washes.  Next would be dehydration with graded ethanol series.  Final dehydration in Propylene Oxide (PO)  then into PO and Resin mixes until finally into pure resin.   I am not sure about 7100 plastic resin, but we use Embed 812 with NMA, DDSA, and DMP-30 as the hardener.  This resin is very common in EM as is very stable under the electron beam.  You can also use LR White resin or another product called Spurrs.  It depends on what you are trying to accomplish and weather it will be labeled or not.  If you need a recipe I can send it to you.

The tissue is the question.  Lung and other tissue pose infiltration problems and you might want to look into microwave and vacuum processing.  We routinely use microwave and vacuum processing for all our tissues.  I find it cuts down on time from three days to six hours.

If you are not set-up for EM processing then you can send it out.  First they should ask you what you want to study in your EM images and then they will tell you how to prepare the tissue to send to them.  The types of organelles and what you want to see in your images will determine how you fix your tissue.

Just as in Histology there are layers of knowledge for the right situation.  It is the same for EM.

Good luck,

Lita Duraine
EM Technologist
Bellen Lab
HHMI- Molecular Genetics
Duncan Neurological Research Institute
1250 Moursund St.
Houston, TX 77030
Rm: N1165.17
MS: N1125.50
832-824-8772 TEM Room
979-549-6526 Cell
http://flypush.imgen.bcm.tmc.edu/lab/people/lita.php

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