Hi Indra.

It may not be the antibody. It can't be. Make every solution fresh for new start.

Regards.

Thank you for the answers, I will try higher dilutions and start everything from the beginning. I have used a positive and negative controls and those samples look fine (not swollen) so I'm hoping its the dilutions.

Thanks again

Indi!

Sounds like your making progress. Its hard to figure out what's really happening without knowing your protocol and how you do it. But, here are some tricks to consider/understand (or to talk to a lab tech/histologist on to help you figure out a modified protocol):

Solution related:

1) Make sure your PBS is made with correct concentrations and that your using the same batch of PBS for ALL samples! You might need to make a fresh solution and measure things out carefully...

2) Put a big bowl over your samples (somehow cover it from the open atmosphere) while they are being fixed or are sitting around during waiting periods.

Sample and/or solution related:

3) Try more incubate-wash steps with Ab at higher conc. for lower time. For waiting periods, don't extend it into the next day. Try getting everything done quick.

4) Do the above steps in #2, but if you have final washes, do a few more than usual for ALL samples. Then, try to image as soon as you can/immediately.

5) Try a different antibody. Maybe your anti-body has physiological/pharmalogical activity. Could its binding trigger receptor activation (i.e., act as agonist) (and, also, cause tonicity changes that lead to swelling?)? If you don't wanna change the Ab, it might help to use a different fixation protocol that's a little stronger (across all samples) as long as no significant artifacts are created (this might reduce overall fluoresence levels though).

In any case, keep at it! I believe in you. :D