[Histonet] PLP fixative

[Gayle Callis]

Our prion people do this routinely .   The PLP is made up fresh just before 
animal euthanasia begins.  PLP perfusion of rodent model via heart (after 
anesthetic) using a tidy little circulating pump followed by 5 hours 
immersion fixation for tissues dissected out post-perfusion.  Each brain 
(mouse, hamster, etc) has a different processing schedule protocol to avoid 
overdehydration.   Brain and spinal cord, other tissues section without 
problems as long as hair and any bone fragments are avoided at dissection. 
To facilitate tongue fixation, it is bisected before immersion.  Brains are 
sliced using a matrix (go to www.myneurolab.com, hope this is correct) for 
precise sectioning, orientation of brain).

IHC for PrP antigens is done with sections are put into formic acid, a form 
of retrieval for this unique PrP. They have excellent results with 
IHC.  PLP fixed tissues are never processed with or after NBF fixed 
tissue.  The researcher is concerned NBF (even a sniff of!) will compromise 
antigen.

Frequently, PLP perfused hamster brain and tongue are cryoprotected with 
30% sucrose and cut at 50 um with dedicated Leica 1850 cryostat using 
antiroll device.


>Cindi,
>
>The recipe for PLP (Periodate-Lysine-Paraformaldehyde) fixative and a sample
>protocol (at least for immuno) using perfusion fixation is on the Chemicon
>website at:
>http://www.chemicon.com/techsupp/Protocol/perfusion.asp
>
>There are a few instances of using PLP with paraffin embedding, so the
>processing should work OK.  Whether you can retrieve reactivity from paraffin
>sections seems to be related to the antigen.  For example, see the NIEHS 
>website:
>http://dir.niehs.nih.gov/dirlep/immuno/protocols.htm
>
>Cheers,
>
>Bob Chiovetti
>
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Gayle Callis
MT,HT,HTL(ASCP)
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610
406 994-6367 (lab with voice mail)
406 994-4303 (FAX)