[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.
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
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
I just wonder then, how do you TRANSFER the block from cryomold onto the
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
Thank you very much for your kindest help!
Yichao WU,Ph.D candidate
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