Ed-Paul Greengard, at Rockefeller, studied phosphorylation state of a
brain protein as it was in living animals, and won a Nobel prize for the
body of work of which this was a part.  Necessary is to use a microwave
fixation apparatus, and fix in less than a second from the living,
un-anaesthetized state.  Please do not try this with a kitchen or
histology microwave not designed for this application, it would take way
too long and get way to bad a result, and be too inhumane.  Appropriate
equipment is costy, and may be found at the following link:
http://www.myneurolab.com/mnl/mnlsite/ViewProduct.asp?idproduct=470101&c
atdesc=Histology+Equipment&subcatdesc=Sacrifice+Equipment&idsubcategory=
21

If the link doesn't work, go to www.myNeuroLab.com, scroll down to under
"Histology", click on "sacrifice equipment".  Click on "Microwave
Fixation".  For mouse brain, the 5kw model would be sufficient.  Contact
me for further information.


Cordially,

Charles W.  Scouten, Ph.D.
myNeuroLab.com
5918 Evergreen Blvd.
St. Louis, MO 63134
Ph: 314 522 0300  
FAX  314 522 0277
cwscouten@myneurolab.com
www.myneurolab.com


-----Original Message-----
From: Wesley Chang [mailto:wesley.chang@probes.com] 
Sent: Thursday, August 29, 2002 2:56 PM
To: HistoNet Server
Subject: RE:phospho-specific-antibody staining

Ed-

I spent a fair amount of time at my last job working with detecting
phosphorylated proteins (mainly p-CREB and p-ERK 1/2) in mouse brain
sections and can share some of what we learned. Our experience was that
perfusion is not the best way to preserve the phosphorylated epitopes,
mainly because (1) the use of an anesthetic, usually pentobarbital,
shuts down brain activity and will affect the pattern of CREB
phosphorylation and (2) the time required to do the perfusion and get
the brain out can also lead to de-phosphorylation artifacts.

After testing several methods, we settled on an acute dissection
procedure that does not use pentobarbital or transcardial perfusion.
Essentially, you approach the tissue with the mindset of a biochemist,
rather than an anatomist: the animal is rapidly sacrificed, the brain is
very rapidly removed and plunged into ice-cold saline solution (or
Ringer's-type buffer), which firms the tissue up a bit and shuts down
enzyme activities. Then, the brain is transferred to a chilled metal
matrix (ASI/Harvard) that has slots every 2mm for making cuts with a
razor blade (you could probably also use a Vibratome). The brain is then
cut into ~5 slices, which are then placed into a vial of chilled
fixative. Assuming your animal use protocol allows you to do this
procedure, you can get the brain out and into fixative in 1-2 minutes
(with some practice). Granted, the tissue morphology is not quite as
good as with perfusion and you end up with a lot of blood in the tissue,
but you will almost certainly get more staining, and I believe the
pattern of staining will be a more accurate "snapshot" of
phospho-proteins.

By the way, using 15 ml/minute for a mouse is likely too high a flow
rate. You run the risk of blowing apart the microvasculature. I don't
know if this may have something to do with the "gradient" of staining
you're seeing.

A couple of notes about the staining: even with the above method, the
overall staining intensity with anti-p-CREB/ERK was not very strong. I
started with fluorescent secondary antibodies for detection, but then
found I could get much better signal using an amplification scheme with
a tertiary antibody coupled to alkaline phosphatase and detecting with
BCIP/NBT. If I were to do this again, I'd probably try using tyramides
(TSA). I'd also recommend trying thinner tissue sections (10-12 um), as
you'll get better antibody penetration and possibly better imaging
(depending on your scope set up). Also, I found that antigen retrieval
(in citrate buffer) greatly increased staining intensity.

Hope this helps. Good luck, and feel free to email with any questions.

Wes

Wesley S. Chang, Ph.D.
Molecular Probes, Inc.
4849 Pitchford Avenue
Eugene, OR 97402-9165
Tel:(541) 984-5675 x542
FAX:(541) 984-5698

Date: 28 Aug 2002 15:31:09 -0500
From: Ed Boyden 
Subject: phospho-specific-antibody staining

Dear All,

I am trying to observe the phosphorylation state of a nuclear protein
(CREB) 
in the brain, in mice. Briefly, I perfuse the animal with formaldehyde, 
cryoprotect in  sucrose, and then I cut 30 um slices and stain with a 
phospho-specific antibody.

The problem is, the staining is very dim in cells that are located a few

hundred microns from the surface of the brain (e.g., cells far away from
the 
edges of each 30 um slice). I've tried 5 non-phospho-specific
antibodies, 
which have no such problem. This suggests that this is a problem specfic
to 
the phosphorylation state of the protein.

Does anyone know if protein phosphorylation state is labile during 
perfusion? I am already doing very fast perfusions with a pump (15
ml/min 
for a mouse), preserving the brain in ice as soon as the blood runs
clear, 
and postfixing. Should I perfuse with phosphatase
inhibitors?

Any suggestions would be greatly appreciated!

Best,
Ed

Ed Boyden
ed_boyden@hotmail.com
Stanford Neuroscience Program
Beckman B103, Stanford, CA 94305
phone (650) 736-1066/fax (650) 725-3958
--