[Histonet] Immunofluorescent staining of IgG on kidney frozen
sections
yichao wu
yichaowu <@t> hotmail.com
Mon Mar 22 10:01:00 CST 2004
Dear Young (What a nice name!:-))
Thank you so much for your kindest help and the important information!And
our director has urged me to order such kind of F(ab')2 antibody for our
immunostaining,when she heard of your suggestions today.
Yes,the usage of IgA F0316 (vs F0204), IgM F0317 (vs F0203) as well as IgG
F031 (vs F0315) is very reasonable for lower background. I agree this
entirely after reading the production explanations at DAKO website.
I just wonder, since F(ab')2 antibodies are inclined to produce lower
background if there is any,should we change all antibodies we used now to
this kind of antibodies if possible?
And I just wonder what is the reason of its low background for the F(ab')2
antibody?:-) Would the Fc part of the primary antibody react with the Fc
receptors on human cells in the sections? However, the primary antibody is
from a different species, would the Fc part of this antibody still react
with Fc receptor on human cells? Another possibility may be, the
completements on the human sections may fix the primary antibody with Fc
part.Still is the different species problem.Maybe there is a kind of
reaction accross different species? (Is my description clear? Sorry for any
confusion)
Besides,could you also advise me how the frozen sections should be blocked?
Now we used 10% Bovine Calf Serum in PBS.
And before the mounting of the frozen sections for inspection under
fluorescent microscope, should the sections be rinsed in some kind of
solution like CuSO4 or NaBO4 to decrease autofluorescence if there is any? I
am afraid that these two kind of solutions would decrease the intensity of
the "real positive"signals.
Thank you very much for all of your help! I know a friend from Australian
Sydney,a very very nice lady.
Connie
Yichao WU
Research Institute of Nephrology
Jinling Hospital
305 East Zhongshan Road
Nanjing 210002
P.R.China
>From: Young Kwun <kwuny <@t> email.cs.nsw.gov.au>
>To: 'yichao wu' <yichaowu <@t> hotmail.com>
>Subject: RE:Renal IF
>Date: Mon, 22 Mar 2004 15:50:54 +1100
>
>Dear Yichao,
>Next time please call me just Young. That's the Australian way (In most
>cases).
>Other FITC antibodies we use Fc as well. IgA( All Dako's, F0316), M
>(F0317), C3 (F0201), CIq(F0254),
>Fibrinogen (F0111) Kappa(F0340) and Lambda(F0341). I normally follow their
>suggested upper dilution. If the dilution they suggest is 1:10 - 1:20, then
>1:20 would be a quite safe bet generally.
>Another thing is that you do not need to fix in aceton or methanol at all
>for renal or skin IF. If you have a know positive case, try to do staining
>without fixation. Unless we are busy, we normally dry the slides about 30
>mins at room temp.
>Feel free to write to me if you have further questions.
>
>Regards,
>
>Young
>
>
>
>-----Original Message-----
>From: yichao wu [SMTP:yichaowu <@t> hotmail.com]
>Sent: Monday, 22 March 2004 14:59
>To: kwuny <@t> email.cs.nsw.gov.au
>Subject: Thank you very much
>
>Dear Dr. Kwun,
>
>Thank you very much for your kindest help! You are the first one who shares
>this experience with me.Thank you, really.
>
>Connie
>
>Yichao WU
>Nanjing China
>
>
> >From: Young Kwun <kwuny <@t> email.cs.nsw.gov.au>
> >To: 'yichao wu' <yichaowu <@t> hotmail.com>,"Histonet (E-mail)"
> ><histonet <@t> pathology.swmed.edu>
> >Subject: RE: [Histonet] Immunofluorescent staining of IgG on kidney
>frozen
> >Date: Mon, 22 Mar 2004 09:37:12 +1100
> >
> >Hi,
> >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
> >Tel)61-2-9767-6075
> >Fax)61-2-9767-8427
> >kwuny <@t> email.cs.nsw.gov.au
> >
> >The information contained in this message is intended for the named
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> >If you are not the named addressee please do not copy, distribute, take
>any
> >action reliant on, or disclose anything in this E-mail message to any
>other
> >person or organisation. If you have received this message in error please
> >delete the email and notify me immediately. Views expressed in this
>message
> >are those of the individual sender and are not necessarily the views of
> >Central Sydney Area Health Service.
> >
> >
> >
> >-----Original Message-----
> >From: yichao wu [SMTP:yichaowu <@t> hotmail.com]
> >Sent: Sunday, 21 March 2004 22:36
> >To: andreah <@t> imclone.com
> >Cc: histonet <@t> lists.utsouthwestern.edu
> >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
> >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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