RE: [Histonet] Immunofluorescence in paraffin sections

From:"Edmondson David (RBV) NHS Christie Tr"

1.  I have no experience of using fluoresence BUT the pretreatments that you use to reveal antigens should be analagous to other detection systems.   SINCE IT IS THE ANTIGENS THAT ARE IMPORTANT.
So IF trypsin is the business then around 0.05% in 0.1% Calcium Chloride, for say ten minutes woks for various antigens by immunoperoxidase methods.     If the fluoresence is a sensitive as one imagines then times and concentrations may be shorter.
   The time that is best is what you find works best rather than what I say.  What about other pretreatments? 
2. Can not help here
Christie Hospital
Manchester UK

-----Original Message-----
From: []On Behalf Of
Sent: 21 March 2005 16:21
Subject: [Histonet] Immunofluorescence in paraffin sections

Hello, all,
I am doing immunofluorescence in paraffin sections. I have some difficulties in it:
1: For pretreatment of tissue sections, I plan to incubate sections with trypsin solution, but I do not know :what kind of concentration should I choose ? (0.01% trypsin, 0.05% or o.1%)how long does it should last? (10min,20min,30 min)
2: Recently, I read a protocol about double immunofluorescence, it shows :Antibodies derived from different animal can be mixed and incubated as a cocktail (example: rabbit anti-A and mouse anti-B). The same is valid for secondary antibodies (example: goat anti-rabbit Texas Red conjugated and goat anti-mouse fluorescein conjugated). but If secondary antibodies cross-react, what should I do? My friend told me that I could use depletion method. but I am not sure.
Can anybody do me a favor?
Thank you!

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