[Histonet] Please help with me--routine immunofluorescent staining on kidney specimens

[yichao wu]

Dear Dr. Yaskovich,

Thank you for your suggestion.But now we prefer to do renal 
immunofluorescent stainings of IgG IgA IgM C3 C4 C1q on frozen sections. I 
have realized that frozen sections are the most frequently applied in other 
labs for renal immunofluorescent stainings of those immunoglobulins.

The difficulty of ours is the pseudo-positive results on acetone-fixed 
paraffin-embedded sections.That is to say,sometime we have positive result 
of IgA on such sections,but on the frozen section of the same patient, it is 
negative! As for other kind of fixatives,formalin is also not suitable for 
immunofluorescent stainings of those immunoglobulins.

The reason for such pseudo-positive results  may involve multi-factors,I 
imagine.

1) Maybe the process of acetone-fixation and paraffin-embedding cause 
auto-fluorescence.
2) Maybe the antibodies we used could not react with those immunoglobulins 
specifically? But what we used is from DAKO.

3) Another information is, on acetone-fixed paraffin-embedded sections,we 
have compared,
a) Direct staining of IgA with FITC conjugated rabbit-anti-human IgA 
antibody
b) Indirect staining of IgA with rabbit anti-human IgA as primary antibody 
first,then FITC-conjugated swine anti-rabbit secondary antibody.
The results are just different!And frequently all positive in glomerular 
capillaries for different degree!
And in some cases the frozen sections of the same patient is negative.

Could anyone kindly give me suggestions? Now we want to do immunofluorescent 
staining of such  immunoglobulins totally on frozen sections.But we do not 
know the most popular protocol of it in renal pathology in details. Any 
suggestion would be helpful to us! And we just could not stand 
pseudo-positive results anymore.

Yichao WU
Jinling Hospital
Nanjing 210002
P.R.China


>From: "Yaskovich, Ruth A (NIH/NIDCR)" <ryaskovich <@t> dir.nidcr.nih.gov>
>To: 'yichao wu' <yichaowu <@t> hotmail.com>
>Subject: RE: [Histonet] Please help with me--routine immunofluorescent 
>staining on kidney specimens
>Date: Thu, 18 Mar 2004 09:47:38 -0500
>
>Can you do it the opposite cut the Snap frozen section first then melt down
>for paraffin much better sections? Acetone/Methonol is not a good fixative
>for Paraffin.
>Ruth Yaskovich
>Neuronal Gene Expression Section
>National Institute of Dental and Crainiofacial Research
>N.I.H.
>-----Original Message-----
>From: yichao wu [mailto:yichaowu <@t> hotmail.com]
>Sent: Thursday, March 18, 2004 8:59 AM
>To: histonet <@t> lists.utsouthwestern.edu
>Cc: tli1 <@t> flowcity.bsd.uchicago.edu
>Subject: [Histonet] Please help with me--routine immunofluorescent
>staining on kidney specimens
>
>
>Dear Dr. Callis, Dr. Li, and everyone here, Hello!
>
>It is a long time no seeing.I am the person who inquired how to prepare
>frozen sections with LN2 last year in June. Later we successfully isolated
>RNA from acetone/methanol-fixed paraffin-embedded sections and then we do
>not pay much attention to frozen sections.
>
>(Ref:
>http://www.histosearch.com/histonet/Jun03A/Aboutquotbetterfrozensect.html)
>
>But now we have encourtered with a most enormous difficulty.Ours are kidney
>biopsy specimens from patients. And We found that immunofluorescent
>stainings of IgG IgA and IgM on acetone/methanol-fixed paraffin-embedded
>sections are not accurate. There are somewhat pseudo-positive results in
>them.Therefore we go on to transform all routine immunofluorescent 
>stainings
>
>from paraffin-embedded sections to snap-frozen sections.
>
>But how is the most popular standard protocol now in the  immunofluorescent
>stainings in renal specimens? Could you kindly give us some suggestions? 
>The
>
>antibodies we used are from DAKO, including rabbit anti-human primary
>antibodies and swine anti-rabbit secondary antibodies.The washing buffer is
>PBS.
>
>Another questions to Dr. Callis, you have mentioned to me that,
>"A cryomold is what you embed tissue in - to form a block.The chuck is what
>holds the block when cutting in cryostat.DO NOT USE THE CHUCK FOR SNAP
>FREEZING..."
>
>I just wonder then, how do you TRANSFER the block from cryomold onto the
>chuck?
>
>The second question is,could I store the frozened biopsy specimens in 
>liquid
>
>N2 till the sectioning? Will it increase the possibility of empty spaces in
>the specimens?
>
>Thank you very much for your kindest help!
>
>Yichao WU,Ph.D candidate
>
>Jinling Hospital
>Nanjing 210002
>P.R.China
>
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