Question:  do you rinse your cells thoroughly with PBS after fixation?

We fix tissue sections with 2% PFA 5 min (cells have been
fixed the same, using cold temps), rinse with PBS x 3 changes
do IFA with TRITC or FITC, and mount with Aquamount, let them stand
overnight before reading the slides.  We found we had fuzzy results if
the slides were read immediately, OR if the PBS was not rinsed off the
back of the slides with water soaked Kimwipes (salts dried on slide)

Also, we are careful to protect from light when incubating with
fluorochromes.  I am sure that is a given technic, sorry to remind
the masses.

Something new on the market, new fluorochromes from Molecular Probes,
these are more stable ie. less photobleaching and are brighter than
fluorescein or lissamine rhodamine. called Alexa 488 (FITC
substitute) and Alexa 546 (TRITC substitute).  WE will be trying them next
week.  Conjugates can be done with their kit (sweet), purchased or
conjugated to strepavidin (our choice).  This could be an answer to
to woes of losing fluorescence with one dose of UV light aka photobleaching.

Hope this helps.

Gayle Callis