AT the request of one who deletes RE: daily digest, a repeat on this
message (I think this may be a third time, the reply came through twice,
sorry, not me doing that!)

Will be more careful to put more specific info in subject line in future,
thanks for reminding me.


>> Zinc fixation is NOT ZINC FORMALIN, often confused because of the way
>> Pharmingen put it in their catalog.
>> 
>> Zinc TRIS Buffer fixation needs to be tested, not large tissues, I suggest
>> a fixation time study, up to optimal time.  If tissue is not fixed by this
>> "fixative" then fixation will continue during paraffin processing.  You
>> will get decent morphology, but sectioning is a bit difficult as tissues
>> are drier, crunchier.  A good ice water soak is necessary, be sure to fix
>> small, thin pieces of tissue and don't overprocess (too long in dehydrants,
>> etc)
>> 
>> You get primary antibody concentrations similar to acetone fixed frozen
>> sections, NO ANTIGEN RETRIEVAL necessary.
>> 
>> You can use whatever Alk phos chromogen you want, DAKO has a nice one
>> called Fuchsin +, a bit more sensitive, red, excellent, we use this with
>> their SA-AP, inhouse dilution. We tried this with Vector BCIP/NBT with good
>> results on murine T cell marker.  Chromogen is what you prefer, red or blue
>> color, with or without hematoxylin counterstain.
>> 
>> Levamisole quenches endogenous alk phos in tissues with exception of gut
>> alk phos, which is quenched by acetic acid (have done this on mouse gut,
>> fixed with Zinc TRIS Buffer, was lovely)
>> 
>> You should access the original articles, Nitta et al, for technic and
>> antibody dilution ranges, was helpful.  Pharmingen catalog has those
>> references.
>> 
>> At 04:25 PM 12/8/00 +0200, you wrote:
>> >Hi
>> >
>> >I need to visualise CD31 in rat tissue. There appears to be only one
>> >monoclonal available from Research Diagnostics. It requires zinc fixation
>> >prior to paraffin embedding and  alk. phos detection.
>> >
>> >Does anyone have an opinion on
>> >1. Zn fixation
>> >2.what is the optimal chromogenic substrate for alk phos
>> >3. Dako mentions an endogenous alk phos inhibitor, levamisole which they
>> >put in some of their kits
>> >
>> >Thanks
>> >
>> >_____________________________________________________
>> >
>> >Nazlia Samodien
>> >
>> >Cardiovascular Research Unit
>> >Dept. of Cardiothoracic Surgery
>> >University of Cape Town Medical School
>> >Anzio Rd, Observatory, 7925, South Africa
>> >
>> >Tel. +27 21 406 6398 (office)
>> >        +27 21 406 6476 (Secretary)
>> >Fax + 27 21 448 5935
>> >
>> >Email: ctssamodien@samiot.uct.ac.za
>> >
>> >> Gayle Callis
>> Veterinary Molecular Biology
>> Montana State University
>> Bozeman MT 59717-3610
>> 406 994-4705
>> 406 994-4303
>> 
>-------------------------------------------------------------
    
> John A. Kiernan,
> Department of Anatomy & Cell Biology,
> The University of Western Ontario,
> LONDON,  Canada  N6A 5C1
>   Phone: (519) 661-2111
>   FAX (Department): (519) 661-3936
>   E-mail: kiernan@uwo.ca
>
>
>
>
Gayle Callis
Veterinary Molecular Biology
Montana State University
Bozeman MT 59717-3610
406 994-4705
406 994-4303