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Help! Sections come off slide during mounting


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#1 soulsista

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Posted 19 September 2012 - 11:11 AM

I am doing immunofluorescence on frozen mouse spleen tissue. Everytime I go to mount the coverslip onto my slide with Vectorshield+DAPI, the tissue moves and seems to disappear. When I look under the microscope, the tissue seems to be smeared, folded and can only see very few nuclei.

Protocol:
The tissue is cryopreserved using concentrations of sucrose from 5%-20%. The tissue is then embedded in OCT in an isopentane bath cooled by liquid nitrogen. The tissue block is cut onto superfrost plus slides and dried at 37C for 30min. The slides are then kept at -20C. When required, the slides are warmed to room temp (~15min) then fixed in cooled acetone for 15min and dried for 10-15min. I then proceed to washing the sections with PBS, block O/N, primary antibody 1hr, wash, secondary antibody 1hr, wash then add coverslip with Vectorshield + DAPI mounting media.

When I do this with heart sections, the section stays in place when putting the coverslip on.

Has anyone experienced this? Should spleen sections be treated differently? Do they have to be dried longer or do I need to coat the slides with something? Any help would be much appreciated.
Thanks.

#2 Curtis

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Posted 20 September 2012 - 12:28 AM

I wonder why it doesn't move in the washing steps or during antibody incubation, and only moves when you mount it!! do you put the slides on a rotator during incubation? if yes, then don't I would also block for 1 hr and incubate with primary antibody O/N. Maybe you need to modify this in your protocol. You can also reduce acetone fixation to 5 min, or use another fixation reagent...I have never used vectashield, I use Invitrogen's ProLong Gold, but it's definitely not from the mounting buffer.




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