Re: phospho-specific-antibody staining

From:Wesley Chang

I've also heard that the microwave fixation method is superior in
capturing the biochemical "state" of brain or other tissues, and should
have mentioned this technology in my reply. My old lab did look into
purchasing one of these units (made in Japan by Muromachi, distributed
by Stoelting), but they're ridiculously expensive (I seem to remember
~$80K), rather out of reach for most non-Rockefeller labs. Still, it is
supposed to be the most rapid way to fix tissues and shut down
biochemical activity, so if your lab is doing lots of these kinds of
studies, it may be worth considering.

Wes Chang

"Charles W. Scouten, Ph.D." wrote:
> 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:
> atdesc=Histology+Equipment&subcatdesc=Sacrifice+Equipment&idsubcategory=
> 21
> If the link doesn't work, go to, 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.
> 5918 Evergreen Blvd.
> St. Louis, MO 63134
> Ph: 314 522 0300
> FAX  314 522 0277
> -----Original Message-----
> From: Wesley Chang []
> 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
> Stanford Neuroscience Program
> Beckman B103, Stanford, CA 94305
> phone (650) 736-1066/fax (650) 725-3958
> --

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