We tried nearly everything for the noradreanline IHC and did't get any
staining. After several calls with the techs of the company, they told
me I've to do a glutaraldehyd fixation, every other fixation will wash
the noradrenaline out of the tissue (even the cryosection did't give a
stain).
Using adrenal glands as positive control, I got a weak positive stain
using the GA-Fixation. But I'm not happy with this, because the
samples I got are FFPE or snap frozen. So what to do to proceed?
Because I don't know any way back through the formalin fixation, I
decided to go on with the snap frozen tissue.
I would be very happy, if someone would tell me: "Hey, thats all
bullshit, you just need the antibody XXX of the company YYY and
verything will work fine with this noradrenaline IHC"
Until this happens, I try with the snap-frozen material, keep my
fingers cross, throw pennies in every fontaine, rub oil-lamps and lion
noses....
You see, I'm desperately needing help,
Frauke
Zitat von anh2...@med.cornell.edu:
Good advice indeed. However, I don't recommend you rinse in water
after sucrose. Sort of defeats the purpose. Instead if you need to
remove excess sucrose rinse in either 50/50 sucrose/OCT or just OCT.
Also, glutaraldehyde fixation usually renders tissue difficult to
immunostain. You have to consider whether your fixation is
appropriate for your antigen/antibody.
-----Original Message-----
From: "Swain, Frances L" <swainfranc...@uams.edu>
Date: Wed, 18 Mar 2009 08:12:45
To: Dr. med. Frauke Neff<ne...@staff.uni-marburg.de>;
histonet@lists.utsouthwestern.edu<histonet@lists.utsouthwestern.edu>
Subject: RE: [Histonet] cryoprotection possible?
Usually the cryoprotection is carried out after the specimens are
fixed and before they are frozen. If you have a sample you can
spare you might try making up some 20% Sucrose placing the frozen
sample in it. putting it in the refrigerator and letting it stay in
the 20% sucrose until it drops to the bottom of the container. You
might have to change the 20% Sucrose a couple of times as you know
sucrose can grow bacteria easily. When the specimen has dropped to
the bottom of the container, remove it, rinse in a couple of changes
of distilled water and quick freeze the sample. Try cutting and
staining the sample and see if the morphology is good if it is then
you can do all of your samples to correct the problem. There should
be no thawing between removal of the sample from the storage to the
cryostat.
Hope this helps
Frances Swain
________________________________________
From: histonet-boun...@lists.utsouthwestern.edu
[histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Dr. med.
Frauke Neff [ne...@staff.uni-marburg.de]
Sent: Wednesday, March 18, 2009 3:47 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] cryoprotection possible?
Dear Histonetters,
I'm supposed to do a noradrenaline ihc on rat brains that have been
snap frozen
and afterwards glutaraldehyd fixed. While the morphology is okay in the
"test"-tissue we used to establish the protocol, it was very poor in
the tissue
of the trial animals.
It appeared mooth eaten and disrupted.
I assume the brains were thawed and refrosted due to moving the
samples between
several cities. Does anyone know a method /a tip how I can save some proper
morphology?
I checked the archive and found sucrose as cryoprotectant, but this
is supposed
to do it while the tissue is frozen or is it possible to use it while the
tissue thaws?
Thank you all in advance for your patience and help,
Frauke
--
Dr. med. Frauke Neff
AG Neurologische Therapieforschung
Neurologie
Philipps-Universität Marburg
Rudolph-Bultmann Str. 8
35039 Marburg
06421/5866304
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