Dear Susan, wich protocol do you use for immunofluorescence in paraffin sections? Do
you have autofluorescence in your sections? Are you using the correct antibodies?

Also you can try brighter secondaries as Alexa Dyes. They work very well in tissues
if you have some residual fluorescence...

Susanna Castel

En/Na "Garfield, Susan (NCI)" ha escrit:

> We are staining paraffin embedded liver sections for desmin using either
> peroxidase or FITC/TxRed.  For some unknown reason, we get beautiful staining
> using peroxidase, but not with the fluorescent secondary (either FITC or TxRed).
> We know that the primary AB is working as expected.  We can do both the
> peroxidase and fluorescent staining in frozen sections and both work great.
> When we try to do the same in paraffin embedded sections, the peroxidase works
> fine, but the fluroescent staining is undetectable (even when we only dilute the
> secondary AB 1:10).  Can anyone suggest a solution to this problem or even what
> the problem could be?
>
> Thanks in advance for your help.
>
> Susan Garfield
> Laboratory of Experimental Carcinogenesis
> Division of Basic Sciences
> National Cancer Institute, NIH
> Building 37, Room 3C28
> 37 Convent Drive MSC4255
> Bethesda, MD  20892-4255
> Phone: 301-496-5688; Fax: 301-496-0734