Immunofluoresece/FISH Protocol - Looking for breakpoints (Jun/06/2007 )

I am currently working on a dual immunofluorescence/ in situ hybridization project. As things run now, it is a three day protocol. The problem I'm having is that I often don't have three consecutive days to work on this project. I'm wondering if there is any way that I could store my slides after I finish the IF and do the in situ after a couple of days. I would appreciate any suggestions.

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to give any hints I think we need your protocol in more detail...

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Sure thing. I didn't want to start out with too much info.

I'm working with spermatogenic cells. I first make a squash prep of a small section of seminiferous tubule, snap freeze the slide in liquid nitrogen, remove the cover slip and fix in an ethanol series (70%, 95% and 100%). The cells are permeabilized overnight at 4C in the 100% ethanol. They are rehydrated in 1x PBS before hybridization with the primary antibody overnight at 4C.

After hybridization, the slides are washed (3x) in 1x PBS and incubated with a fluorescent secondary antibody for 30 min at Room Temp

After washing (again 3x in 1x PBS), the slides are hybridized with a 22-mer DIG-labeled oligo probe for the message of interest (overnight at 37C). The following morning the slides are washed as follows:

1x with 50% formamide in 2x SSC (15 min, 37C)
1x with 2x SSC (15 min, 37C)
1x with 1x SSC (15 min, Room Temp)
1x with 4x SSC (1-2 min, Room Temp) to equilibrate cells

Cells are incubated with hyb mix containing anti-DIG for 20 min-1 hour. Slides are washed (10 min, RT) in 4x SSC, 4x SSC w/ 0.1% Triton-X and 4x SSC.

The cells are then incubated with a fluorescent labeled secondary antibody and washed as above.

They are then dried and mounted with Vectashield containing DAPI.

My major questions are:
Should I do anything to fix my fist signal before proceeding to FISH?
Can I store my slides between the immuno-fluorescence and the FISH?
If so, how should I store them?

Thanks for any input.

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