Can you watch someone else in the lab do the fixing and staining? They may be adding the methanol in a more gentle way or using -20 degree methanol or something different like that. It might be something straight forward like that unless your cells are overly confluent at the time of fixation (they can peel off in a sheet or partial monolayer) or you are using a 293 cell-derivative (293T, HEK293) to do your assay since they adhere poorly.

Dear Mr. Morello

Thanks for your attention. It is not possible for me to watch them currently.

But you mentioned some useful points here.

I have not used cold methanol, I will try it this time.

I will seed less cells to reduce cell confluency at the day of infection.

What is your idea of using some other options for fixation and staining?

Hello Nasrin,

I guess your cells are not getting fixed properly. Also may I ask what overlay media are you using? Try aspirating the overlay before adding the methanol and I would say increase the time of fixation. Add methanol gently along the side/walls of the well.

In our lab for plaque assay we use 10% Formalin for fixing the cells for a minimum of 20mins to an hour, and later use crystal violet for staining and it works great every time. You could try doing that. Also we typically seed 2E5 Vero cells/well in a 24 well dish for infections.

Good luck, hope it helps!

I fixed and stained too like Jagaree Das. I used an agarose overlay since that was easy to prepare and use and would add concentrated buffered formalin on top of the agarose overlay (final conc. >4% formalin), let it diffuse through and fix for a couple hours, and then remove the overlay and formalin by aspiration with a wide-bore aspirator. I've used methylcellulose too but always removed it before formalin fixing as above.