RE: [Histonet] Immunofluorescent staining of IgG on kidney frozen

From:Young Kwun

We use IgG F(ab)2 (Dako F0315) instead of Ig fraction you are using. We 
follow manufacturer's suggested dilution which is 1:10  without any problem 
with the background staining.
We used to use another IgG Fc in 1:50 dilution from a company called 
DiaSorin in USA. It was a very good antibody. But we were unable to get 
their antibodies anymore for some reason.
I hope this helps.

Young Kwun, PhD
Senior Hospital Scientist
Dept. of Anatomical Pathology
Concord Hospital
Concord NSW 2139 Australia

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-----Original Message-----
From:	yichao wu []
Sent:	Sunday, 21 March 2004 22:36
Subject:	RE: [Histonet] Immunofluorescent staining of IgG on kidney frozen

Thank you for your reply!It is very helpful to me.I have once thought of 
problem too.

Since the background of IgA and IgM is very low and they are serum
immunoglobulins too,it seems strange for the strong background of IgG.

Mr.George Cole advised me that the concentration (1:50 in dilution) for the 
anti-human IgG antibody may be too high.However, our tech told me that
further dilution would prevent those positive signals from being present.

Hence I just wonder how other departments of renal diseases handle the
immunofluorescent staining in biopsy specimens? Please kindly give me some
hints since it is terrible for the diagnosis of patients with bad
immunofluorescent staining results.It really hurts :-(

Thank you very much in advance!

Yichao WU,Ph.D Candidate
Research Institute of Nephrology
Jinling Hospital
305 East Zhongshan Road
Nanjing 210002

>Subject: RE: [Histonet] Immunofluorescent staining of IgG on kidney frozen
>Date: Sat, 20 Mar 2004 13:07:18 -0500
>I expect it is not background but real signal from all the IgG in the
>tissue. IgG is normally at high concentration in mouse tissue and will 
>staining especially in a blood filled organ like the kidney.
>----- Message from "yichao wu"  on Sat, 20 Mar 2004
>17:25:27 +0800 -----
>Subject:    [Histonet] Immunofluorescent staining of IgG on kidney frozen
>sections---High background!
>Dear Dr.Callis,Dr.Yaskovich,,Dr. Li,and all histonetters,
>Thank you very much for all of your reply.The frozen sections we make now
>have better mophology with Liquid N2 freezing.
>Still three questions with regarding to kidney immunofluorescent staining.
>1) We stain human IgG on kidney frozen sections with antibody from DAKO
>(F0202),and dilution is 1:50,but the background is always strong.
>Other antibodies to IgA(F0204 from DAKO),IgM(F0203 DAKO),C3(F0201
>DAKO),C4(F0169 DAKO)&C1q (F0254) all produce dark background under
>fluorescent microscope,which is highly preferred.
>Why IgG is so strange? Any problem with the antibody? Or the unspecific
>staining for IgG is special?
>2) What kind of O.C.T is the best?
>Thank you very much!
>Yichao WU
>Research Institute of Nephrology
>Jinling Hospital
>Nanjing 210002

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