Dear Jeyanthi,

I have generally obtained better results using 4% paraformaldehyde solution (10 mins) followed by triton 0,2% permeabilization (for IF) vs metanol-acetone fixation. In either case, make sure to wash thoroughly in order to remove the fixative.  

Cell confluence, antibody (if using IF for FFU) and washes (if using crystal violet for PFU) can influence the resolution of the foci. 

Also, the quality (high viral titers) of your virus and an established kinetic of plaque formation (kinetics can be faster for FFU vs PFU) will correlate with better results. 

In the link, I provide a good reference for an overview of alternative methods if your FFU assay continue to give you an hard time.

Good luck,

Nicolas

https://www.labome.com/method/Methods-for-Rapid-Virus-Identification-and-Quantification.html

Thank you very much Dr. Nicolas. That;s very helpful. I guess I have found an FFU assay expert here. I would like to take this opportunity to ask another question. I had hands-on FFU assay for the first time and realized after substrate development, tiny spots seems to be floating which I believe are the foci. But why are they floating and can be washed away? I tried to figure out the reason but didn't find the answer.  Have you experienced this? I'm using HRP conjugated anti mouse IgG  as the secondary antibody (1: 250)and metal enhanced DAB substrate (1:100).

Hi Jeyanthi,

the floating foci that you are seeing might be detached/unattached cells. Do you see wholes or empty spaces in the background indicating that cells were washed away?

You could try to treat you plate with collagen or polylysine to minimize cell detachment. 

Nicolas

http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0043960