[Histonet] IF-protocol on skin

[Gudrun Lang]

Hi all,

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

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

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