[Histonet] RE: C4d antibody on Paraffin sections

[yichao wu]

Dear Donovan,

Sorry for the late reply. Actually I learned the method from a refence,but I 
could not remember the exact journal and article now.

The sections I used were acetone-methanol(1:1) fixed paraffin embedded 
sections with. I have not tried on formalin-fixed paraffin-embedded 
sections.But I remember that in the reference mentioned above,it reads that 
this method is applicable on formalin-fixed sections.

The procedures are not special,just add the pretreatment with 88% formic 
acid.That is to say,
Dewaxing;
Draw a circle around the sections with PAP pen;
Incubate with PBS;
Incubate with 88% formic acid (diluted with ddH2O i.e. pure water) for 20 
minutes in room temperature;
Incubate with 10% fetal bovine serum as the blocker (or other kinds of block 
serum);
Incubate with monoclonal mouse anti-C4d antibody (A213 from Quidel USA) at 
4C overnight;
Incubate with FITC-conjugated rabbit anti-mouse IgG antibody (from DAKO) for 
40 mins at RT;
Observe under fluorescent microscope...

Besides, it is not any advertisement for those products I mentioned! :-) 
Just a little experience.

Wish it is helpful. And thank you for your attention.You are much welcomed!

Yichao WU,Ph.D Candidate

Research Insititute of Nephrology
305 East Zhongshan Road
Nanjing 210002,P.R.China






>From: "Donovan, Mark" <M.Donovan <@t> alfred.org.au>
>To: "'yichaowu <@t> hotmail.com'" <yichaowu <@t> hotmail.com>
>Subject: C4d antibody on Paraffin sections
>Date: Tue, 30 Mar 2004 15:39:03 +1000
>
>Dear Yichao,
>
>I read your response to Carmen Loiselle regarding the Quidel antibody to 
>C4d
>with great interest.
>
>Could you provide me with more details of your protocol for using the
>antibody on formalin fixed paraffin embedded sections.
>
>I have used this antibody on frozen sections but have not had any success 
>in
>getting it to work on formalin fixed material. Your advice would be very
>much appreciated.
>
>Regards,
>
>Mark Donovan
>Immunohistochemistry Laboratory
>Anatomical Pathology
>The Alfred Hospital
>Melbourne, Australia
>
>
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