[Histonet] Immunofluorescence in paraffin sections

[Edmondson David (RBV) NHS Christie Tr]

Hello,
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?=20
2. Can not help here
Dave
Histology
Christie Hospital
Manchester UK

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu =
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu]On Behalf Of =
pex0220 <@t> yahoo.com.cn
Sent: 21 March 2005 16:21
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] Immunofluorescence in paraffin sections


Hello, all,
=20
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.
=20
Can anybody do me a favor?
Thank you!
=20
Guofeng
=20



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