[Histonet] IF-protocol on skin

Gudrun Lang gu.lang <@t> gmx.at
Sun Feb 8 12:29:25 CST 2009

Hi all,

I’d like to compare my immunofluorescence protocol with other experienced

Here is my protocol. Please give me your comments, if there’s a hidden

Are the antibody-titers ok?


1.	freeze the nativ skin-specimen in the cryostat with OCT
2.	let wait in a -20°C freezer until beginning (1-3 days), wrapped in 
3.	cut at 6-8 microns, airdry for 30-60 min
4.	30 min fitc-labelled anti-IgG (Zymed, 1:20 and 1:40), IgM (Dako,1:10
and 1:20) , 
IgA (Dako, 1:10 and 1:20), Complement3c (Dako 1:10 and 1:20), Fibrinogen
(Dako 1:10 and 1:20)
all diluted in PBS
5.	rinse in PBS 2x 5 min
6.	coverslip with antifading medium


Last week we had a positiv skin with an IgG-line on basement membran. Ok,
that’s fine. Then we tried an IHC with our usual protocol for IgG, IgM and
IgA on the Benchmark XT. We expected to see a beautiful red band of IgG in
the FFPE-skin. But - The result was a very intense staining with all
Immunoglobulins in the skinblister, but also in the dermis, very intense at
the basmentmembran. What’s that? Has anyone tried IHC instead of IF?


Curious Gudrun

More information about the Histonet mailing list