smFISH basic questions

[j.z...@gmail.com]

Hello,
I just started using the smFISH method on E.coli cells and have a few questions:

* My main issue is a problem of auto-fluorescence of the sample (even prior to staining, so not due to probe specificity). I've tried increasing the formamide concentration for the washes - this helps a bit but not enough...any suggestions on the matter?

* Also, the cells sometimes clump together in bunches (they are still distinct cells, just together in groups). I am using ETOH (final concentration 70%) to permeablize the cells. I take care to do this step gradually (first DDW and then the ETOH- in 3 separate portions), because I know this is a delicate step and can cause clogging if not done gradually. Is there something else I can do to help the clumping? (the fixation is done in 2% Paraformaldehyde in PBS, incubation for an hour, washes, centrifugation and addition of ETOH for permeabilization, gradually).

* Also, I've seen that many protocols (but not all) use gel pads to take images in the microscope. I searched all over the web and could not find why? Does this make the cells more stable and less prone to move between the glass slide and the cover?

Thanks in advance!
Jonathan