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

[yichao wu]

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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