Hi all,
I've been trying to get an IF for neutrophils (using an anti-CD66b antibody) in FFPE liver tissue sections to work, but no success so far. The protocol is run on a Ventana automated system. I got the same antibody to work perfectly on IHC in the same kind of tissue sections, but I've been having issues when trying IF, even though the tissue goes through the same antigen retrieval process as for IHC. Any input as to what might be going wrong for the IF that prevents me from getting specific staining?
Thank you!
Joao
Hi,
do you have sufficient controls?
For example: Is the reaction you see in IHC really specific (what happens at "secondary-only)? Do you have a positive control for your IF secondary? Do you work light-protected?
Hi Vivica,
The reaction I see in IHC is specific, the secondary-only doesn't have any noticeable unspecific staining. In IF, the secondary-only shows some unspecific staining, which look similar to when the primary is added, hence the dificulty in identifying what's real. And yes, I work light-protected. What positive control are you referring to?
Thanks for your reply.
João
Using IF with FFPE tissue is usually not recommended, since FFPE tissue will have a fair bit of autofluorescence, which can lead to the impression of non-specific staining that you are getting. Not sure why you're doing IF if you have a working IHC protocol though.
What I mean by "positive control for secondary ab": You might have a problem concerning the correct dilution of the secondary antibody. Or that combination is less sensitive than your IHC protocol. Therefore you should incubate another primary antibody targetting a high abundance antigen followed by your secondary to check if it works in general.
Another possibility (which depends on the host species of your antibodies): Try to detect the fluorescenct secondary by a (enzyme-labelled) tertiary antibody against its host species.
By the way: Do you use a mounting medium compatible with IF (aqueous based, containing anti-fade)?
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