[Histonet] Immunofluorescent staining of IgG on kidney frozen

[yichao wu]

Dear LuAnn,

Thank you for your suggestions.And George Cole has also suggest that to 
me.Thank you all.

Yichao


>From: LuAnn Anderson <ander093 <@t> tc.umn.edu>
>To: "yichao wu" <yichaowu <@t> hotmail.com>
>Subject: RE: [Histonet] Immunofluorescent staining of IgG on kidney  frozen
>Date: Mon, 22 Mar 2004 10:21:44 -0600
>
>Hello Yichao,
>You will need to find a known positive control and titer the IgG. Start at 
>1:50 and increase 1:100, 1:200, 1:400, 1:800, etc (checkerboard). Then 
>screen them and determine which titration works best for low background but 
>positive staining. You could do dilutions in between if you think 
>necessary. 1:50 sound much too high, which would produce the background you 
>are seeing.
>
>
>
>
>
>At 07:35 PM 3/21/04 +0800, you wrote:
>>Thank you for your reply!It is very helpful to me.I have once thought of 
>>the 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
>>P.R.China
>>
>>>From: andreah <@t> imclone.com
>>>To: yichaowu <@t> hotmail.com
>>>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 
>>>give
>>>staining especially in a blood filled organ like the kidney.
>>>
>>>----- Message from "yichao wu" <yichaowu <@t> hotmail.com> on Sat, 20 Mar 2004
>>>17:25:27 +0800 -----
>>>
>>>To:   histonet <@t> lists.utsouthwestern.edu, tli1 <@t> flowcity.bsd.uchicago.edu
>>>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
>>>P.R.China
>>
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