[Histonet] Help with immunofluorescence

[Dolors Fuster]

We are trying to demonstrate SHH and Survivin in human embrionic
colo-rectal tissue by Immunofluorescence in FFPE samples.

We are using:

   * goat polyclonal SHH (N-19)  sc-1194 with donkey anti-goat
   secondary sc-2024
   * mouse monoclonal Survivin (D-8) sc-17779 with donkey anti-mouse
   secondary sc-2099

As antigen retrieval we have used saponin 0,1% in PBS in some slides 
and  a post-fixation in carnoy in others.

Carnoy postfixed slides seem to shoe a better image. However,and 
specially in Sonic, we are  seeing  that some cells in the submucosa 
appear labeled while epitelial cells in the mucosa remain unlabeled

As we are seing these images in samples treated only with the conjugated 
Ab but also in samples with primary and conjugated, we think it could be 
a cross or unespecific reaction of the secondary Ab or may be  the 
primary has not
reacted properly.

May you help us at this point?

We would also ask you for some suggestion about tissue controls for 
these Ab.

Any advice would be appreciated.

Thanks in advance


Dolors Fuster
Tècnic Especialista en Anatomia Patològica i Citologia
Departament d'Anatomia i Embriologia Humana
Facultat de Medicina (HCP)
Universitat de Barcelona