[Histonet] Re: HELP PLEASE/alk. phosph.

[C.M. van der Loos]

Hi,
I am missing something here? Why would you perform Fast Blue staining first and then embed in paraffin and have them next stained immunohistochemically with several markers???
Using New Fuchsin kit (DakoCyto) you will obtain a more permanent AP reaction product. It just dissolves a little bit in alcohol. Furthermore, there is Vector Red AP chromogen kit from Vector Labs. Its reaction product is also dissolving a little bit in alcohol. If you have strong staining from your immuno there is no problem processing the slides through alcohol-xylene and mount up organically.
If you change your marker enzyme to b-galactosidase and using X-gal as substrate/ferri-ferrocyanide as chromogen one yields a reaction product that can stand everything without being affected or dissolved.
Hope this helps

Chris van der Loos, PhD
Dept. of Pathology
Academic Medical Center
Amsterdam - The Netherlands


----- Original Message ----- 
>From  Bruce Abaloz <brucea <@t> unimelb.edu.au> 
Date  Tue, 27 Apr 2004 11:29:49 +1000 
To  histonet-bounces <@t> lists.utsouthwestern.edu, histonet <@t> lists.utsouthwestern.edu 
Subject  [Histonet] Re: HELP PLEASE(John/Gayle/Connie/Cynthia/Patsy/Lee & Peggy/Tony, SOMEONE....) 
Hi, my name is Danielle & I need some advice PLEASE -
I have been using alkaline phosphatase histochemistry to detect primordial germ cells in PFA-fixed marsupial embryos. I use 1mg/ml Fast Blue salt and 1mg/ml Naphthol AS phosphate sodium salt in 0.2M Tris buffer, pH 9.4. The staining is good but I have been told that it is not permanent. Ideally, I would like to dehydrate these stained specimens, embed them in paraffin and then section and stain them immunohistochemically for other germ cell markers and several growth factors. Does anyone have a protocol utilising alkaline phosphatase histochemistry that would allow me to do this? Thanks for any advice in advance,

Dr Danielle Hickford
Research Fellow
Department of Zoology, University of Melbourne,
Australia 3010.>hickford <@t> unimelb.edu.au