Hi all, Id like to compare my immunofluorescence protocol with other experienced histotechs.
Here is my protocol. Please give me your comments, if theres 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, thats 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. Whats that? Has anyone tried IHC instead of IF? Curious Gudrun _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
